Neuronal organization of the large-cell segment of the cat's red nucleus]

When studying the neuronal organization of the large-cell part of the red nucleus in cats by the Golgi and Golgi-Kopsch methods three types of neurons have been revealed: large (50-90 mu), medium-sized (20-50 mu) and small cells (8-20 mu). long axon and short-axon neurons were found as well as long-dendrite cells with few thorns (the length of the dendrites form 600-900 mu) and short-dendrite cells (up to 400 mu). On basis ofanalysis of neuronal groups found in the large-cell part of the red nucleus of the cat a neuronal map of this part of the central nervous sytem is composed.     

Red and Processed Meat Intake Is Associated with Higher Gastric Cancer Risk: A Meta-Analysis of Epidemiological Observational Studies

Background

Red and processed meat was concluded as a limited-suggestive risk factor of gastric cancer by the World Cancer Research Fund. However, recent epidemiological studies have yielded inconclusive results.

Methods

We searched Medline, EMBASE, and the Cochrane Library from their inception to April 2013 for both cohort and case-control studies which assessed the association between red and/or processed meat intake and gastric cancer risk. Study-specific relative risk estimates were polled by random-effect or fixed-effect models.

Results

Twelve cohort and thirty case-control studies were included in the meta-analysis. Significant associations were found between both red (RR: 1.45, 95% CI: 1.22–1.73) and processed (RR: 1.45, 95% CI: 1.26–1.65) meat intake and gastric cancer risk generally. Positive findings were also existed in the items of beef (RR: 1.28, 95% CI: 1.04–1.57), bacon (RR: 1.37, 95% CI: 1.17–1.61), ham (RR: 1.44, 95% CI: 1.00–2.06), and sausage (RR: 1.33, 95% CI: 1.16–1.52). When conducted by study design, the association was significant in case-control studies (RR: 1.63, 95% CI: 1.33–1.99) but not in cohort studies (RR: 1.02, 95% CI: 0.90–1.17) for red meat. Increased relative risks were seen in high-quality, adenocarcinoma, cardia and European-population studies for red meat. And most subgroup analysis confirmed the significant association between processed meat intake and gastric cancer risk.

Conclusions

Our findings indicate that consumption of red and/or processed meat contributes to increased gastric cancer risk. However, further investigation is needed to confirm the association, especially for red meat.     

Annexin 6 immunoreactivity in select cell populations in the rat brain.

We examined the distribution of annexin 6 (ANX6) in rat brain with immunohistochemistry (IHC). Several neuronal cell populations were intensely labeled with the ANX6 monoclonal antibody (MAb), including layer 5 of neocortex, the lateral septum, the lateral hypothalamic area, the red nucleus, and the Purkinje cell layer in cerebellum. Neuronal immunolabeling was localized to the nucleus and the cytosol. Darkly stained ANX6-immunoreactive glia, with the morphology characteristic of astrocytes, were abundant in the hippocampus, substantia nigra reticulata, and cerebellum. Evidence suggests that ANX6 may function in neuronal and glial calcium-dependent processes.     

Decreased Release of D-Aspartate in the Guinea Pig Spinal Cord After Lesions of the Red Nucleus

This study attempts to determine if fibers that project from the guinea pig red nucleus to the spinal cord use L-glutamate and/or L-aspartate as transmitters. Unilateral injections of kainic acid were placed stereotaxically in the red nucleus to destroy the cells of origin of the rubrospinal tract. Six days after the injection, Nissl-stained sections through the lesion site showed that the majority of neurons in the red nucleus ipsilateral to the kainic acid injection were destroyed. In addition, the lesioned area included parts of the surrounding midbrain reticular formation. Silver-impregnated, transverse sections of the cervical spinal cord revealed the presence of degenerating fibers contralaterally in laminae IV-VII of the gray matter. Ipsilaterally, very sparse degeneration was evident in laminae VII and VIII of the gray matter. Two to six days after surgery, the electrically evoked, Ca2(+)-dependent release of both D-[3H]aspartate, a marker for glutamatergic/aspartatergic neurons, and gamma-amino[14C]-butyric acid ([14C]GABA) was measured in dissected quadrants of the spinal cervical enlargement. Lesions centered on the red nucleus depressed the release of D-[3H]aspartate by 25-45% in dorsal and ventral quadrants of the cervical enlargement contralaterally. The release of [14C]GABA was depressed by 27% in contralateral ventral quadrants. To assess the contribution of rubro- versus reticulospinal fibers to the deficits in amino acid release, unilateral injections of kainic acid were placed stereotaxically in the midbrain reticular formation lateral to the red nucleus. Nissl-stained sections through the midbrain revealed the presence of extensive neuronal loss in the midbrain and rostral pontine reticular formation, whereas neurons in the red nucleus remained undamaged. In the spinal cord, degenerating axons were present ipsilaterally in laminae VII and VIII of the gray matter. Some fiber degeneration was also evident contralaterally in laminae V and VI of the gray matter. This lesion did not affect the release of either D-[3H]aspartate or [14C]GABA in the spinal cord. The substantial decrements in D-[3H]aspartate release following red nucleus lesions suggests that the synaptic endings of rubrospinal fibers mediate the release of D-[3H]aspartate in the spinal cord. Therefore, these fibers may be glutamatergic and/or aspartatergic. Because other evidence suggests that rubrospinal neurons are probably not GABAergic, the depression of [14C]GABA release probably reflects changes in the activity of spinal interneurons following the loss of rubrospinal input.     

Responses of neurons of the solitary tract nucleus to noxious colorectal distension in rats

In experiments on anaesthetized rats, the neuronal mechanisms underlying processing of the nociceptive information from the colon within the nucleus of the solitary tract were studied. In addition, the role of nitric oxide in these processes was estimated. Analysis of changes in c-fos expression revealed that nociceptive colorectal distension (CRD) resulted in activation of neurons mainly in the medial, commissural, parvicellular and dorsomedial subnuclei of the solitary tract nucleus. Non-noxious CRD evoked in these subdivisions weak phasic excitatory neuronal responses. Under noxious CRD, neurons with phasic (58%) and tonic (42%) responses were revealed. The phasic neuron responses were significantly enhanced in comparison with non-noxious CRD. Inhibition of the neuronal NO-syntheses resulted in significant decrease of neuron responses to noxious CRD and the number of cells with tonic reactions. Therefore, neurons with tonic responses may be directly related to NO-depended processing ofnociceptive information from colon.     

Quantitative changes in the total proteins of the cells of the hypothalamic supraoptic nucleus after extreme exposures: acute immobilization and deprivation of the paradoxical sleep phase

It has been shown that 1-hour acute immobilization of rats resulted in a decrease of total protein content (per 1 cell) in neurones (by 17% in cytoplasm and 22% in nucleus) of the supraoptic nucleus of the hypothalamus and in their glial satellite cells (by 10%), mainly at the expense of the nuclei of the latter. These changes remained at the same level in the neuronal cytoplasm and satellite cells within 6 hours after cessation of acute immobilization, being abolished in the neuronal nuclei. Deprivation of REM sleep for 24 hours affected these parameters to the same extent as acute immobilization. However, 6-hour rest was sufficient for normalisation in satellite cells and for decrease in protein deficiency in the neuronal nuclei; yet protein deficiency still sustained in the neuronal cytoplasm. REM sleep deprivation was also accompanied by a decrease in tritium incorporation into satellite cell proteins (by 18%); after 3-hour rest, incorporation of the label into satellite cells was restored to the initial level being increased by 20% in neuronal nuclei; another 3-hour rest resulted in normalisation of labeling in the neuronal nuclei and in 27% increase of labeling in the satellite cells.     

Effects of forebrain microinjection of cholecystokinin on dopamine cell firing rate.

In anesthetized rats, midbrain dopamine (DA) neuronal firing rate was differentially sensitive to focal brain microinjection of cholecystokinin peptides (CCK-4 and CCK-8) and N-methyl-D-aspartate (NMDA) into nucleus accumbens, amygdala and prefrontal cortex. Whereas changes in DA neuronal firing rate were frequently observed in response to intra-amygdalar microinjection of CCK peptides, NMDA was most effective in eliciting changes in DA neuronal activity following intra-accumbal microinjection. Thus, stimulation of amygdalar CCK receptors and accumbal excitatory amino acid receptors may participate in the afferent regulation of midbrain DA neuronal function.     

Endocytic vesicles and surface invaginations in cultured vascular endothelium: a morphometric comparison

Ruthenium red staining of plasma membrane glycoproteins of confluent cultured arterial endothelial cells revealed that the limiting membrane of many apparently discrete cytoplasmic vesicles was continuous with the plasmalemma. Surface invaginations accessible to ruthenium red appeared as vesicles when sectioned out of the plane of attachment to the cell surface, Morphometric analysis of ruthenium red-positive (RR+) and ruthenium red-negative vesicles (RR-) indicated that 47.2% of the total apparent vesicle population was RR+ and that those infoldings accounted for 19.6 +/- 1.4% of the cell surface in transverse sections. Whereas 14.9% of the true vesicles (ruthenium red-negative) were coated vesicles, only 1.1% of RR+ "vesicles" were coated pits. These studies show that although many deep infoldings of the cell surface may be misinterpreted as vesicles, almost all are uncoated. The existence of discrete coated vesicles (independent of coated pits) in vascular endothelium in vitro is readily apparent.     

Recurrence plots of neuronal spike trains

The recently developed qualitative method of diagnosis of dynamical systems — recurrence plots has been applied to the analysis of dynamics of neuronal spike trains recorded from cerebellum and red nucleus of anesthetized cats. Recurrence plots revealed robust and common changes in the similarity structure of interspike interval sequences as well as significant deviations from randomness in serial ordering of intervals. Recurring episodes of alike, quasi-deterministic firing patterns suggest the spontaneous modulation of the dynamical complexity of the trajectories of observed neurons. These modulations are associated with changing dynamical properties of a neuronal spike-train-generating system. Their existence is compatible with the information processing paradigm of attractor neural networks.     

Development of human cerebellar nuclei. Morphometric study

The morphometric development of the human cerebellar nuclei was examined in 9 fetuses (16-40 weeks of gestation; WG), an infant (2 months old) and 2 adults (16 and 63 years old). With the morphological observation of serial sections of the brain containing the cerebellar nuclei, the authors measured sections to get several morphometric parameters: the volume of nuclear column and number, packing density and cell body area of neurons. Each nucleus (dentate, emboliform, globose and fastigial nucleus) was recognized even at 16 WG. Nerve cells containing Nissl bodies were observed in all nuclei after 23 WG. Degenerative changes were detected in some neurons for every nucleus at 21 and 23 WG. Three stages were observed in the developmental course of nuclear volume and neuronal packing density: the primary or undifferentiated stage at 16 WG, the secondary stage with variability at 21-32 WG and the tertiary stage with monotonous increase (nuclear volume) or gradual decrease (neuronal packing density) after 35 WG. No significant correlation between neuronal number and gestational age was noticed for every nucleus. The analysis of cell body area (neuronal size) demonstrated that the dentate neurons developed after the intermediate or fastigial neurons. It is concluded that there is a critical period between slightly before 20 WG and slightly after 30 WG, matched with the secondary stage in the development of the cerebellar nuclei.     

Neonatal monosodium glutamate treatment abolishes both delta opioid receptor-induced and alpha-2 adrenoceptor-mediated gastroprotection in the lower brainstem in rats.

Neonatal monosodium glutamate treatment reduced immunoreactive beta-endorphin content in the mediobasal hypothalamus by 50% in adult, male Wistar rats as compared to hypertonic saline-treated littermates; there was also a moderate (approx. 25%) reduction in the rostral part of the nucleus of the solitary tract. In sham-treated adults the intracisternally injected alpha-2 adenoceptor stimulant clonidine (0.47 nmol/rat) and the delta opioid receptor type agonist (D-Ala(2), D-Leu(5))-enkephalin (0.8 nmol/rat) reduced acidified ethanol-induced mucosal lesions in the stomach by 84.1 and 77.5%, respectively, whereas the same doses were completely ineffective in rats treated neonatally by monosodium glutamate. The data taken together with the results of previous studies with the same substances in rats with retroarcuate knife cuts suggest that neuronal damage in the nucleus of the solitary tract region rather than in the arcuate nucleus is responsible for the changes seen in the pharmacological responsiveness.     

Fractionation of nuclei from brain by zonal centrifugation and a study of the ribonucleic acid polymerase activity in the various classes of nuclei

1. The nuclei of the cells of the whole rat brain have been fractionated in a B-XIV zonal rotor with a discontinuous gradient of sucrose. Five fractions were obtained. Zone (I) contained neuronal nuclei (70%) and astrocytic nuclei (23%). Zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (15%). Zone (III) contained astrocytic nuclei (84%) and oligodendrocytic nuclei (15%). Zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained only oligodendrocytic nuclei. 2. The content of DNA, RNA and protein per nucleus was determined for each zone. Although the amount of DNA per nucleus is constant (7pg) the RNA varies from 4.5 to 2.5pg/nucleus and the protein from 38 to 17.6pg/nucleus. The neuronal nuclei have the greatest amounts of protein. The oligodendrocytic nuclei have the least content of RNA and protein. 3. The effects of pH, ionic strength, and Mg(2+) and Mn(2+) concentration on the activity of the nuclear system for synthesis in vitro of RNA have been investigated for unfractionated nuclei. From these studies a standard set of conditions for the assay of nuclear RNA polymerase has been established. 4. The activity of the RNA polymerase in each of the zonal fractions has been determined in the presence and in the absence of alpha-amanitin. Zone (II) is the most active, followed by zone (I). The nuclei of zones (IV) and (V) have comparable activity, which is 40% of that of zone (II). 5. The extent of incorporation of each of the four labelled nucleoside triphosphates by the nuclei from each zone has been measured. These values have been used to calculate the base composition of the RNA synthesized in vitro in each class of nucleus. 6. The effect of changes in the condition of assay of RNA polymerase in the different classes of nuclei has been investigated. Significant differences in the response to concentrations of metal ions and ammonium sulphate have been observed. 7. Homopolymer formation in each zone of brain nuclei has been determined. The extent of formation of the four homopolymers roughly parallels the RNA polymerase activity.     

Evidence for a possible glycinergic inhibitory neurotransmission in the midbrain and rostral pons of the rat studied by gephyrin

The data on the glycinergic transmission in the rostral brainstem are both few and controversial. The present report provides evidence for a possible glycinergic transmission in Sprague-Dawley rats, based on observations of immunocytochemical labeling for gephyrin, a 93 kDa protein and a component of the functional glycine receptor. A monoclonal antibody against gephyrin was used, and the reaction product was visualized by means of avidin-biotin-peroxidase procedure. The reaction product in midbrain and rostral pons was found in neuronal perikarya and in proximal dendrites but in some cases the most distal dendritic branches were also labeled. The neuropil usually displayed a moderate staining with finely granulated reaction product. The most significant immunocytochemical signal was mainly encountered in large and medium-sized neuronal populations of the motor cranial nerve nuclei (III, IV, V), in the reticular formation (laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus, deep mesencephalic nucleus), in the red nucleus, in the intermediate and deep gray strata of the superior colliculus. Only in the substantia nigra and the inferior colliculus the parvocellular cell populations were mainly labeled. The present data suggest a significant inhibitory glycinergic neurotransmission in the rostral brainstem, probably mediated by interneurons.     

Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method

Endo-polygalacturonase (endo-PGase) activity determinations generally rely on viscosity changes or reducing sugar ends produced by this activity over polygalacturonic acid. Torres and coworkers [Enzyme Microb. Technol. 48 (2011) 123–128] showed that ruthenium red (RR) is useful for endo-PGase determination. In this article, we present a high-throughput liquid-based endo-PGase assay based on the RR method and compare it with the viscosity determination method. The reduced assay uses a small volume of enzyme solution, 40 μg of polygalacturonic acid, and 45 μg of RR for each sample determination. Furthermore, we obtained an interconversion factor for RR and viscosity activities.     

Differential co-expression of AMPA receptor subunits in substance P receptor-containing neurons of basal forebrain regions of C57/BL mice

We are interested in cellular co-expression patterns of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptor subunits 1-4 (GluR1-4) in substance P receptor (SPR)-containing neurons of the basal forebrain, which may act as a morphological basis for interaction between neurokinins and glutamate-driven neuronal signaling and excitotoxicity. Immunohistochemistry and laser scanning confocal microscopy in adult C57/BL mice revealed that distribution of SPR-positive neurons overlapped with that of GluR1-4-containing ones in most basal forebrain regions, i.e. the medial septal nucleus, nucleus of diagonal band of Broca, magnocellular preoptic nucleus and substantia innominata. Neurons showing both SPR and GluR1-4-immunoreactivities were found in above cholinergic neurons-rich containing basal forebrain regions. Semi-quantification analysis indicated that about 57-95% of SPR-positive neurons displayed GluR1-4-immunoreactivity. The percentages of AMPA receptor subunits co-localizing in SPR-positive neurons were GluR4 (48%), GluR1 (47%), GluR2 (26%) and GluR3 (20%), respectively. However, the neurons co-expressing SPR and GluR1-4 were hardly detected in the basal nucleus of Meynert of the basal forebrain. The co-localization of SPR and AMPA receptors has provided a molecular basis for functional interaction between neurokinins and AMPA receptors-mediated signaling in basal forebrain neurons. This study has also implied that glutamate-driven neuronal transmission and excitotoxicity can be modulated by neurokinin peptides in most basal forebrain regions but not in the basal nucleus of Meynert, suggesting that neurokinins or SP may play certain roles in determining neuronal functional properties or excitotoxic susceptibility in the various basal forebrain regions of mammals.     

Improved staining of negative binding sites with ruthenium red on cryosections of frozen cells

Hexavalent cationic dye ruthenium red (RR) binds to anionic sites of cellular components, predominantly to the surface coat rich in glycoconjugates, and can be used as a marker of negative binding sites. Due to limited penetration of RR only superficial layers of cells are stained satisfactorily. To improve RR staining of L1210 leukemic cells isolated from culture and concentrated by centrifugation, cryosections of frozen cells were treated by RR to expose simultaneously all the cells and their components to the dye treatment. Cells were fixed with 2% glutaraldehyde in cacodylate buffer (CB), soaked in 2.2 mol/l sucrose and frozen by plunging into liquid nitrogen. Ultrathin cryosections were cut at a temperature of -90 degrees C, transferred to Formvar coated copper grids, postfixed with 1% OsO4 and stained with 0.05% RR in CB for 60-120 min. After removing RR solution with filter the grids were dried and examined electron microscopically. The resulting staining was a combination of a negative contrast (the plasma membrane and membranes of intracellular organelles) and of a positive contrast (cytoplasmic matrix and the extracellular coat). RR staining of negative binding sites on cryosections has proved useful for uniform exposure of all cells and cellular compartments to the dye and especially of external coat containing glycoconjugates.     

Effects of different spectral lights on <Emphasis Type="Italic">Oncidium</Emphasis> PLBs induction,proliferation, and plant regeneration

The effects of different spectral light distribution on in vitro induction and proliferation of Oncidium protocorm-like bodies (PLBs) and subsequent growth of plantlets were investigated. Shoot tips (5 mm in length) of proliferating shoots of Oncidium “Gower Ramsey” were vertically incubated on 1/2 Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ 6-benzyladenine (BA), and grown under either monochromatic red light-emitting diodes (LEDs) (RR), blue LEDs (BB), yellow LEDs (YY) or green LEDs (GG). Cultures grown under fluorescent lamps (FL) were used as control. Selected FL-induced PLBs were cut into 3- to 4-mm sections and incubated on MS medium supplemented with 1.0 mg l⁻¹ BA and 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), and grown under RR, BB, YY, GG, or FL. Moreover, FL-differented shoots (15 mm in length with two leaves) were incubated on 1/2 MS medium with 0.5 mg l⁻¹ NAA, and grown under either FL, RR, 10% blue + 90% red LEDs (1BR), 20% blue + 80% red LEDs (2BR), 30% blue + 70% red LEDs (3BR), BB, 80% red + 10% blue + 10% far-red LEDs (RBFr), or 80% red + 10% blue + 10% green LEDs (RBG). Overall, the red light spectrum enhanced induction, proliferation, and the carbohydrate contents of PLBs, as well as subsequent plantlet lengths, while the blue spectrum promoted differentiation, protein accumulation, and enzyme activities in PLBs, as well as pigment content accumulation in PLBs and developing plantlets. The combination of red and blue LEDs resulted in higher energy efficiency as well as dry weight and enzyme activities in these plantlets.     

Fermentable carbohydrate alters hypothalamic neuronal activity and protects against the obesogenic environment

Obesity has become a major global health problem. Recently, attention has focused on the benefits of fermentable carbohydrates on modulating metabolism. Here, we take a system approach to investigate the physiological effects of supplementation with oligofructose-enriched inulin (In). We hypothesize that supplementation with this fermentable carbohydrate will not only lead to changes in body weight and composition, but also to modulation in neuronal activation in the hypothalamus. Male C57BL/6 mice were maintained on a normal chow diet (control) or a high fat (HF) diet supplemented with either oligofructose-enriched In or corn starch (Cs) for 9 weeks. Compared to HF+Cs diet, In supplementation led to significant reduction in average daily weight gain (mean ± s.e.m.: 0.19 ± 0.01 g vs. 0.26 ± 0.02 g, P < 0.01), total body adiposity (24.9 ± 1.2% vs. 30.7 ± 1.4%, P < 0.01), and lowered liver fat content (11.7 ± 1.7% vs. 23.8 ± 3.4%, P < 0.01). Significant changes were also observed in fecal bacterial distribution, with increases in both Bifidobacteria and Lactobacillius and a significant increase in short chain fatty acids (SCFA). Using manganese-enhanced MRI (MEMRI), we observed a significant increase in neuronal activation within the arcuate nucleus (ARC) of animals that received In supplementation compared to those fed HF+Cs diet. In conclusion, we have demonstrated for the first time, in the same animal, a wide range of beneficial metabolic effects following supplementation of a HF diet with oligofructose-enriched In, as well as significant changes in hypothalamic neuronal activity.     

Regulation of NMDA receptor subunits and nitric oxide synthase expression during cocaine withdrawal

The present study characterized the effects of withdrawal from cocaine on the expression of NMDA receptor subunits (NR1, NR2B) and neuronal nitric oxide synthase. FosB induction was measured to confirm that repeated cocaine exposure influenced protein expression, as previously reported. Administration of cocaine followed by 24 h, 72 h, or 14 days of withdrawal resulted in alterations of NR1 and NR2B subunits and neuronal nitric oxide synthase expression as measured by immunohistochemical labeling of rat brain sections. Optical density analyses revealed significant up-regulation of NR1 in the ventral tegmental area at 72 h and 14 days of withdrawal. Structure-specific and withdrawal time-dependent alterations in NR2B expression were also found. After 24 h of withdrawal, cocaine-induced decreases in NR2B expression were observed in the nucleus accumbens shell, whereas increases in NR2B expression were found in medial cortical areas. Two weeks of withdrawal from cocaine caused an approximately 50% increase in NR2B subunit expression in regions of the cortex, neostriatum, and nucleus accumbens. In contrast, cocaine-induced up-regulation of neuronal nitric oxide synthase was transient and evident in cortical areas only at 24 h after the last drug injection. The results suggest that region-specific changes in interactions among proteins associated with the NMDA receptor complex may underlie neuronal adaptations following repeated cocaine administration.     

Age-Correlated Loss of Dopaminergic Binding Sites in Human Basal Ganglia

Abstract: Human caudate nucleus, putamen, substantia nigra, and nucleus accumbens were analyzed for the effects of age on dopaminergic binding sites. Decreases in the number of dopaminergic binding sites were detected with age in caudate nucleus (44 specimens from three sample groups) and substantia nigra (n = 12). In caudate nucleus, the decline in [³H]2-amino-6,7-dehydroxy-1,2,3,4-tetrahydronaphthalene sites was three times greater than for [³H]spiperone, but age changes were significant in only two of the three sampling groups. No age changes in binding were detected in the putamen (n = 44) or nucleus accumbens. Age, sex, and tissue source all significantly contributed to variance. However, cause of death, time from death to tissue freezing, and length of storage did not influence dopaminergic binding in the caudate nucleus or putamen. Relative to the life-span, the age-correlated decrease in dopaminergic binding sites of human brain approximates that in aging rodent striatum. Comparisons of altered dopaminergic binding with other age-correlated changes suggest that neuronal loss may not be involved in the loss of binding sites before midlife.