Density Of Immunolabeling Research Articles

Microglial and Astrocytic Responses in the Human Midcingulate Cortex in Huntington's Disease.

Patients with Huntington's disease can present with variable difficulties of motor functioning, mood, and cognition. Neurodegeneration occurs in the anterior cingulate cortex of some patients with Huntington's disease and is linked to the presentation of mood symptomatology. Neuroinflammation, perpetrated by activated microglia and astrocytes, has been reported in Huntington's disease and may contribute to disease progression and presentation. This study sought to quantify the density of mutant huntingtin protein and neuroinflammatory glial changes in the midcingulate cortex of postmortem patients with Huntington's disease and determine if either correlates with the presentation of mood, motor, or mixed symptomatology. Free-floating immunohistochemistry quantified 1C2 immunolabeling density as an indicative marker of mutant huntingtin protein, and protein and morphological markers of astrocyte (EAAT2, Cx43, and GFAP), and microglial (Iba1 and HLA-DP/DQ/DR) activation. Relationships among the level of microglial activation, mutant huntingtin burden, and case characteristics were explored using correlative analysis. We report alterations in activated microglia number and morphology in the midcingulate cortex of Huntington's disease cases with predominant mood symptomatology. An increased proportion of activated microglia was observed in the midcingulate of all Huntington's disease cases and positively correlated with 1C2 burden. Alterations in the astrocytic glutamate transporter EAAT2 were observed in the midcingulate cortex of patients associated with mood symptoms. This study presents pathological changes in microglia and astrocytes in the midcingulate cortex in Huntington's disease, which coincide with mood symptom presentation. These findings further the understanding of neuroinflammation in Huntington's disease, a necessary step for developing inflammation-targeted therapeutics. ANN NEUROL 2023;94:895-910.

Thyroid hormones, Sertoli cell proliferation and differentiation in progenies from carbamazepine-treated rat dams during pregnancy and lactation.

Carbamazepine (CBZ) is used in the control of seizure and affective disorders, causing hypothyroidism. Thyroid hormones regulate the Sertoli cell proliferation and differentiation. Clinical aspects must be considered since epileptic fertile women need to continuously use CBZ during pregnancy and lactation. This study aimed to evaluate the effects of CBZ on testis development of rat offspring from dams treated during pregnancy/lactation. Rat dams received CBZ (20mgkg-1 day-1 ) or vehicle by intra-peritoneal route during gestation and lactation. Progenies were euthanised at 4, 14, 41, 63 and 93-days post-partum (dpp) for the evaluation of T3, T4 and TSH plasma total levels. Testicular cross sections were submitted to anti-Ki67, anti-PCNA, anti-p27kip1 and anti-transferrin immunolabelling for the evaluation of Sertoli cells. There was a significant reduction in p27kip1 -positive Sertoli cell numerical densities and an increase in TSH level at 14 dpp. CBZ exposure affected the volume density of transferrin-positive immunolabelling at 63 dpp. These results suggest that CBZ may cause a dysregulation of the controller system of thyroid hormones homeostasis leading to an increase in the proliferation rate at the neonatal phase and a differentiation delay of the Sertoli cell, culminating in an altered function at late puberty. The occurrence of hypothyroidism cannot be completely discarded.

Abnormalities in the copper transporter CTR1 in postmortem hippocampus in schizophrenia: A subregion and laminar analysis

Dysbindin-1 modulates copper transport, which is crucial for cellular homeostasis. Several brain regions implicated in schizophrenia exhibit decreased levels of dysbindin-1, which may affect copper homeostasis therein. Our recent study showed decreased levels of dysbindin-1, the copper transporter-1 (CTR1) and copper in the substantia nigra in schizophrenia, providing the first evidence of disrupted copper transport in schizophrenia. In the present study, we hypothesized that there would be lower levels of dysbindin-1 and CTR1 in the hippocampus in schizophrenia versus a comparison group. Using semi-quantitative immunohistochemistry for dysbindin1 and CTR1, we measured the optical density in a layer specific fashion in the hippocampus and entorhinal cortex in ten subjects with schizophrenia and ten comparison subjects. Both regions were richly immunolabeled for CTR1 and dysbindin1 in both groups. In the superficial layers of the entorhinal cortex, CTR1 immunolabeled neuropil and cells showed lower optical density values in patients versus the comparison group. In the molecular layer of the dentate gyrus, patients had higher optical density values of CTR1 versus the comparison group. The density and distribution of dysbindin-1 immunolabeling was similar between groups. These laminar specific alterations of CTR1 in schizophrenia suggest abnormal copper transport in those locations.

Spinalization and locomotor training differentially affect muscarinic acetylcholine receptor type 2 abutting on α-motoneurons innervating the ankle extensor and flexor muscles.

Complete thoracic spinal cord transection (SCT) impairs excitatory cholinergic inputs to ankle extensor (soleus; Sol) but not to flexor (tibialis anterior; TA) α-motoneurons (MNs) modifiable by locomotor training applied post-transection. The purpose of this study was to investigate whether Sol and TA MNs adapt to changes in cholinergic environment by differential regulation of their muscarinic receptors M2 (M2R). We examined Chrm2 (M2R gene) transcript level, high-affinity 3-quinuclidinyl benzilate-3 H ([3 H]QNB) ligand binding, distribution and density of M2R immunolabeling in lumbar (L) segments in intact and SCT rats, with or without inclusion of 5-week treadmill locomotor training. We show that at the second week after SCT the levels of Chrm2 transcript are reduced in the L3-6 segments, with [3 H]QNB binding decreased selectively in the L5-6 segments, where ankle extensor MNs are predominantly located. At 5weeks after SCT, [3 H]QNB binding differences between the L3-4 and L5-6 segments are maintained, accompanied by higher density of M2R immunolabeling in the plasma membrane and cytoplasm of TA than Sol MNs and by enriched synaptic versus extrasynaptic M2R pools (52% TA vs. 25% Sol MNs). Training normalized M2R in TA MNs, improved locomotion, and reduced frequency of clonic episodes. Our findings indicate higher sensitivity of TA than Sol MNs to cholinergic signaling after SCT, which might shorten flexor twitches duration and contribute to generation of clonic movements. Synaptic enrichment in M2R density may reflect a compensatory mechanism activated in TA and Sol MNs to different extent in response to reduced strength of cholinergic signaling to each MN pool. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Camkii-Mediated Phosphorylation Regulates Distributions of Syngap-α1 and –α2 at the Postsynaptic Density

SynGAP, a protein abundant at the postsynaptic density (PSD) of glutamatergic neurons, is known to modulate synaptic strength by regulating the incorporation of AMPA receptors at the synapse. Two isoforms of SynGAP, α1 and α2, which differ in their C-termini, have opposing effects on synaptic strength. In the present study, antibodies specific for SynGAP-α1 and SynGAP-α2 are used to compare the distribution patterns of the two isoforms at the postsynaptic density (PSD) under basal and excitatory conditions. Western immunoblotting shows enrichment of both isoforms in PSD fractions isolated from adult rat brain. Immunogold electron microscopy of rat hippocampal neuronal cultures shows similar distribution of both isoforms at the PSD, with a high density of immunolabel within the PSD core under basal conditions. Application of NMDA promotes movement of SynGAP-α1 as well as SynGAP-α2 out of the PSD core. In isolated PSDs both isoforms of SynGAP can be phosphorylated upon activation of the endogenous CaMKII. Application of tatCN21, a cell-penetrating inhibitor of CaMKII, to hippocampal neuronal cultures blocks NMDA-induced redistribution of SynGAP-α1 and SynGAP-α2. Thus CaMKII activation promotes the removal of two distinct C-terminal SynGAP variants from the PSD.

Chronic desipramine treatment alters tyrosine hydroxylase but not norepinephrine transporter immunoreactivity in norepinephrine axons in the rat prefrontal cortex

Pharmacological blockade of norepinephrine (NE) reuptake is clinically effective in treating several mental disorders. Drugs that bind to the NE transporter (NET) alter both protein levels and activity of NET and also the catecholamine synthetic enzyme tyrosine hydroxylase (TH). We examined the rat prefrontal cortex (PFC) by electron microscopy to determine whether the density and subcellular distribution of immunolabelling for NET and co-localization of NET with TH within individual NE axons were altered by chronic treatment with the selective NE uptake inhibitor desipramine (DMI). Following DMI treatment (21 d, 15 mg/kg.d), NET-immunoreactive (ir) axons were significantly less likely to co-localize TH. This finding is consistent with reports of reduced TH levels and activity in the locus coeruleus after chronic DMI and indicates a reduction of NE synthetic capacity in the PFC. Measures of NET expression and membrane localization, including the number of NET-ir profiles per tissue area sampled, the number of gold particles per NET-ir profile area, and the proportion of gold particles associated with the plasma membrane, were similar in DMI- and vehicle-treated rats. These findings were verified using two different antibodies directed against distinct epitopes of the NET protein. The results suggest that chronic DMI treatment does not reduce NET expression within individual NE axons in vivo or induce an overall translocation of NET protein away from the plasma membrane in the PFC as measured by ultrastructural immunogold labelling. Our findings encourage consideration of possible post-translational mechanisms for regulating NET activity in antidepressant-induced modulation of NE clearance.

Immunocytochemical analysis of the subcellular distribution of ferritin in Imperata cylindrica (L.) Raeuschel, an iron hyperaccumulator plant

Ferritin is of interest at the structural and functional level not only as storage for iron, a critical element, but also as a means to prevent cell damage produced by oxidative stress. The main objective of this work was to confirm by immunocytochemistry the presence and the subcellular distribution of the ferritin detected by Mösbauer spectroscopy in Imperata cylindrica, a plant which accumulates large amounts of iron. The localization of ferritin was performed in epidermal, parenchymal and vascular tissues of shoots and leaves of I. cylindrica. The highest density of immunolabeling in shoots appeared in the intracellular space of cell tissues, near the cell walls and in the cytoplasm. In leaves, ferritin was detected in the proximity of the dense network of the middle lamella of cell walls, following a similar path to that observed in shoots. Immunolabeling was also localized in chloroplasts. The abundance of immunogold labelling in mitochondria for I. cylindrica was rather low, probably because the study dealt with tissues from old plants. These results further expand the localization of ferritin in cell components other than chloroplasts and mitochondria in plants.

Chronic stress alters the density and morphology of microglia in a subset of stress-responsive brain regions

The current study, in parallel experiments, evaluated the impact of chronic psychological stress on physiological and behavioural measures, and on the activation status of microglia in 15 stress-responsive brain regions. Rats were subjected, for 14 days, to two 30 min sessions of restraint per day, applied at random times each day. In one experiment the effects of stress on sucrose preference, weight gain, core body temperature, and struggling behaviour during restraint, were determined. In the second experiment we used immunohistochemistry to investigate stress-induced changes in ionized calcium-binding adaptor molecule-1 (Iba1), a marker constitutively expressed by microglia, and major histocompatibility complex-II (MHC-II), a marker often expressed on activated microglia, in a total of 15 stress-responsive nuclei. We also investigated cellular proliferation in these regions using Ki67 immunolabelling, to check for the possibility of microglial proliferation. Collectively, the results we obtained showed that chronic stress induced a significant increase in anhedonia, a decrease in weight gain across the entire observation period, a significant elevation in core body temperature during restraint, and a progressive decrease in struggling behaviour within and over sessions. With regard to microglial activation, chronic stress induced a significant increase in the density of Iba1 immunolabelling (nine of 15 regions) and the number of Iba1-positive cells (eight of 15 regions). Within the regions that exhibited an increased number of Iba1-positive cells after chronic stress, we found no evidence of a between group difference in the number of MHC-II or Ki67 positive cells. In summary, these results clearly demonstrate that chronic stress selectively increases the number of microglia in certain stress-sensitive brain regions, and also causes a marked transition of microglia from a ramified-resting state to a non-resting state. These findings are consistent with the view that microglial activation could play an important role in controlling and/or adapting to stress.

Glucose transporter (GLUT-1) distribution in the brain vessels of the adult Italian wall lizard, Podarcis sicula

The GLUT-1 isoform of the glucose transporter is commonly accepted as a reliable molecular marker of blood-brain barrier endothelia in neural vasculature organized in a three-dimensional network of single vessels. The brain of the lizard Podarcis sicula is characterized by a vascular architecture based on a pattern of paired vessels. The presence and distribution of GLUT-1 were studied in adult lizards using both light and transmission electron microscopic techniques. Immunoperoxidase histochemistry was applied to sections from paraffin-embedded brain using gold-conjugated secondary antibodies to localize this antigen on ultrathin sections. The transverse sectioned pairs of vessels did not show the same size and, in particular, the two elements of the same pair often differed in their diameters. Light microscopy revealed immunopositivity in both parenchymal and meningeal vessels. In each transverse-sectioned vascular pair, one element was intensely labelled, and the adjacent one showed only slight or negligible reaction. Colloidal gold particles were restricted to endothelial cells, showing an asymmetric labelling pattern, which was always characterized by markedly higher density of immunolabelling of the abluminal rather than the luminal plasmalemma. Moreover, in every vascular pair, one profile had lower amounts of scantier labelling by gold particles than the adjacent element. This pattern indicates functional differences between the adjacent vascular limbs regarding glucose transport.

Glutamate and GABA immunocytochemical electron microscopy in the hippocampal dentate gyrus of normal and genetic absence epilepsy rats

It is generally accepted that absence epilepsy results from the impairment of GABAergic and glutamatergic neurotransmission. In particular, besides excessive GABA mediation within the thalamo-cortico-thalamic circuit in absence epilepsy, neuronal networks of the hippocampus have recently received attention. In the present study, we examined the density of glutamate and GABA neurotransmitter immunolabeling in the dentate gyrus of the hippocampus of genetic absence epilepsy rats from Strasbourg (GAERS) compared to the control group. GABA and glutamate were found to exist in synaptic vesicles of the mossy fiber terminals of the control and GAERS groups. The density of glutamate immunolabeling within the mossy fiber terminals in the hilar region of GAERS hippocampus was found to be significantly decreased compared to the control group. There was no difference in the density of immunolabeling within GABA nerve terminals between GAERS and control group. The findings of this study suggest that mechanisms underlying absence seizures in GAERS may also manifest themselves in other brain regions such as the hippocampus. The presence of GABA within synaptic vesicles of mossy fiber terminals, as revealed by high resolution ultrastructural immunocytochemistry, has provided additional evidence to the possible modulatory role of GABA on synaptic transmission between the mossy fiber and the target cell.

L-dopa-induced reversal in striatal glutamate following partial depletion of nigrostriatal dopamine with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

We have reported that 1 month following acute (20mg/kg×4) or subchronic (30mg/kg/day×7d) administration of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, there is an increase or decrease, respectively, in the extracellular level of striatal glutamate as determined by in vivo microdialysis [Robinson S, Freeman P, Moore C, Touchon JC, Krentz L, Meshul CK (2003) Acute and subchronic MPTP administration differentially affects striatal glutamate synaptic function. Exp Neurol 180:73–86]. The goal of this study was to determine the effects of treatment with l-dopa (15mg/kg) for 21 days on striatal glutamate starting on day 8 after the first dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine was administered to mice. Following acute administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the increase in extracellular striatal glutamate due to lesion of the nigrostriatal pathway was completely reversed to a level below that found in the vehicle-treated group after l-dopa treatment. Subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment resulted in a decrease in striatal extracellular glutamate that was reversed to the level close to that observed in the vehicle-treated group. There was no change in the density of nerve terminal glutamate immunolabeling associated with the synaptic vesicle pool, suggesting that the alterations in extracellular glutamate most likely originated from the calcium-independent pool. There was a similar decrease in the relative density of tyrosine hydroxylase immunolabeling, a marker for dopamine terminals, within the dorsolateral striatum in both the acute and subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated groups that had been administered l-dopa. There was a decrease in the relative density of immunolabeling within the dorsolateral striatum for the glutamate transporter, GLT-1, following acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment in the groups administered either vehicle or l-dopa. There was no change in GLT-1 immunolabeling following subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. The results demonstrate that the reversal in the extracellular level of striatal glutamate following l-dopa treatment in both the acute and subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated groups is not due to changes in either striatal dopamine nerve terminals or in the density of the glutamate transporter, GLT-1.

Time-dependent changes in extracellular glutamate in the rat dorsolateral striatum following a single cocaine injection

Acute cocaine administration has been shown to alter dorsal striatal plasticity [Proc Natl Acad Sci USA 87 (1990) 6912; Brain Res Bull 30 (1993) 173] and produce long-term neurochemical changes [Pharmacol Biochem Behav 27 (1987) 533]. To date, the effects of acute cocaine on extracellular glutamate and nerve terminal glutamate immunolabeling in the rat dorsolateral striatum have not been reported. To investigate cocaine-induced changes in extracellular glutamate, in vivo microdialysis was carried out in the dorsolateral striatum of rats 1–14 days after receiving a single injection of either vehicle or 15mg/kg cocaine. There was an increase in the group injected with cocaine 1 day prior to measuring extracellular glutamate as compared with the control group. The group injected with cocaine 3 days prior to the microdialysis session had decreased extracellular glutamate levels. Furthermore, extracellular glutamate remained attenuated 14 days after acute cocaine treatment. Striatal glutamate decreased in the cocaine-treated rats after calcium removal, suggesting that cocaine-induced changes in extracellular glutamate were partially calcium-dependent. The density of nerve terminal glutamate immunolabeling was measured using immunogold electron microscopy in the contralateral striatum of the same rats that had been acutely treated with cocaine or vehicle. There were no changes in the density of glutamate immunolabeling within identified nerve terminals making an asymmetrical (excitatory) synaptic contact 1, 2, 3, or 14 days after acute cocaine exposure as compared with the control groups. Hence, these alterations in extracellular glutamate did not result from changes in glutamate immunolabeling within the synaptic vesicle pool. In addition, no changes in glutamate immunolabeling were found in rats that received cocaine 2 h previously or were withdrawn after 1 week of cocaine administration. The results demonstrate that a single injection of cocaine produces biphasic, time-dependent changes in extracellular glutamate in the rat dorsolateral striatum.

High and low responders to novelty show differential effects in striatal glutamate

The goal of this study was to determine whether there was a difference in glutamate within the dorsolateral striatum in mice exhibiting either a high (HR) or low (LR) locomotor response to a novel environment. The number of line crossings over a 30-min-period when the mice were placed in a novel environment was determined, and those mice for which the values were above the mean were in the HR group and those with the values below the mean were in the LR group. In vivo microdialysis was carried out to determine the basal extracellular level of striatal glutamate, and the contralateral striatum was taken to measure the density of glutamate immunolabeling within nerve terminals making an asymmetrical (excitatory) synaptic contact using quantitative immuno-gold electron microscopy. There was a statistically significant difference (35%) in the basal extracellular level of striatal glutamate between the HR and LR groups, with the HR group having a lower level, compared with that of the LR group. There was a 25% difference in the density of nerve terminal glutamate immuno-gold labeling associated with the synaptic vesicle pool in the HR, compared with that in the LR group, but this difference was not statistically significant. There was no change in the basal extracellular level of striatal dopamine between the two groups, but there was a statistically significant difference (73%) in the basal turnover ratio of striatal dopamine and its metabolites in the HR, compared with that in the LR group. The data suggests that the difference in extracellular striatal glutamate between the HR and LR groups is not due to an alteration in basal extracellular dopamine but could be due to an increase in dopamine turnover.

Substance P and neutral endopeptidase in development of acute respiratory distress syndrome following fire smoke inhalation.

To characterize the tachykininergic effects in fire smoke (FS)-induced acute respiratory distress syndrome (ARDS), we designed a series of studies in rats. Initially, 20 min of FS inhalation induced a significant increase of substance P (SP) in bronchoalveolar lavage fluid (BALF) at 1 h and persisted for 24 h after insult. Conversely, FS disrupted 51.4, 55.6, 46.3, and 43.0% enzymatic activity of neutral endopeptidase (NEP, a primary hydrolyzing enzyme for SP) 1, 6, 12, and 24 h after insult, respectively. Immunolabeling density of NEP in the airway epithelium largely disappeared 1 h after insult due to acute cell damage and shedding. These changes were also accompanied by extensive influx of albumin and granulocytes/lymphocytes in BALF. Furthermore, levels of BALF SP and tissue NEP activity dose dependently increased and decreased, respectively, following 0, low (10 min), and high (20 min) levels of FS inhalation. However, neither the time-course nor the dose-response study observed a significant change in the highest affinity neurokinin-1 receptor (NK-1R) for SP. Finally, treatment (10 mg/kg im) with SR-140333B, an NK-1R antagonist, significantly prevented 20-min FS-induced hypoxemia and pulmonary edema 24 h after insult. Further examination indicated that SR-140333B (1.0 or 10.0 mg/kg im) fully abolished early (1 h) plasma extravasation following FS. Collectively, these findings suggest that a combination of sustained SP and NEP inactivity induces an exaggerated neurogenic inflammation mediated by NK-1R, which may lead to an uncontrolled influx of protein-rich edema fluid and cells into the alveoli as a consequence of increased vascular permeability.

Tachykinin substance P depletion by capsaicin exacerbates inflammatory response to sidestream cigarette smoke in rats.

To evaluate the role of substance P (SP)-containing C-fiber nerves in the development of the inflammatory responses to sidestream cigarette smoke (SSCS), female Fischer 344 rats were randomly assigned into vehicle and capsaicin groups, respectively. Then, half the number in each group (N = 24) was nose-only exposed to air or 0.4 mg/m3 total particulate matter of SSCS for 4 h/day for 7 days. Exposure of the vehicle rats to SSCS induced obvious pulmonary neurogenic inflammation as indicated by elevations in plasma extravasation and proinflammatory cytokine secretions [interieukin (IL)-1beta and IL-12]. In addition, except for SP release, SSCS exposure significantly induced the tachykininergic toxicities at the gene level: upregulation of beta-preprotachykinin-I (beta-PPT-I) mRNA. However, neither SSCS exposure nor capsaicin pretreatment affects the immunolabeling density of neurokinin-1 receptor (NK-1R) in airway epithelium. SSCS also significantly inactivated pulmonary neutral endopeptidase (NEP) in lung tissue. Moreover, pretreatment with capsaicin significantly exacerbated the SSCS-induced inflammatory responses mentioned above as well as the release of plasma protein. Considering that capsaicin did not affect the normal control baselines of these parameters except for a decrease in NK-1R mRNA, we conclude that the degree of SSCS-induced inflammatory response was exacerbated because of the depletion of stored SP and/or inactivation of capsaicin-sensitive C-fiber nerves. Our data suggest the loss of afferent tachykinin SP signaling may lead to dysfunction of the sensory C-fiber nerve reflexes during exposure to SSCS, suggesting that SP serves a protective role.

Pretectal and tectal afferents to the dorsal lateral geniculate nucleus of the turtle: an electron microscopic axon tracing and gamma-aminobutyric acid immunocytochemical study.

The pretectal and tectal projections to the dorsal lateral geniculate nucleus (GLd) of two species of turtle (Emys orbicularis and Testudo horsfieldi) were examined under the electron microscope by using axonal tracing techniques (horseradish peroxidase or biotinylated dextran amine) and postembedding gamma-aminobutyric acid (GABA) immunocytochemistry. After injection of tracer into the pretectum, two types of axon terminals were identified as those of pretectogeniculate pathways. Both contained pleomorphic synaptic vesicles and were more numerous in the inner part of the nucleus. They could be distinguished on the bases of size and shape of their synaptic vesicles, type of synaptic contact, and level of GABA immunoreactivity. One type had a higher density of immunolabeling and established symmetric synaptic contacts, whereas the other, less densely immunolabeled, made asymmetric synaptic contacts. In both cases, synaptic contacts were mainly with relay cells and occasionally with interneurons. We suggest that these two types of pretectogeniculate terminals originate in two separate pretectal nuclei. After injection of tracer into the optic tectum, a single population of GABA-immunonegative tracer-labeled terminals was identified as belonging to the tectogeniculate pathway. These were small, had smooth contours, contained very small round synaptic vesicles, and established asymmetric synaptic contacts with long active zones, predominantly with relay cells and less frequently with interneurons, in the inner part of the nucleus. In addition, a population of GABA-negative and occasionally GABA-positive terminals, labeled by tracer injected into either the pretectum or the tectum, was identified as retinal terminals; these were presumably labeled by the retrograde transport of tracer in collateral branches of visual fibers innervating both the GLd and the pretectum or tectum. Comparison of the present ultrastructural findings in turtles with those previously reported in mammals shows that the cytological features, synaptic morphology, and immunochemical properties of the pretectogeniculate and tectogeniculate terminals of both groups share many similarities. Nevertheless, the postsynaptic targets of these two categories of terminals display some pronounced differences between the two groups, which are discussed in terms of their possible functional significance.

Mu-opioid receptor knockout prevents changes in delta-opioid receptor trafficking induced by chronic inflammatory pain

Previous studies from our laboratory have demonstrated that both chronic inflammatory pain, induced by intraplantar injection of complete Freund's adjuvant (CFA), and prolonged (48 h) stimulation of mu-opioid receptors (μOR) by systemic administration of a variety of selective agonists, resulted in enhanced plasma membrane targeting of delta-opioid receptors (δOR) in neurons of the dorsal spinal cord. To determine whether δOR trafficking induced by chronic inflammation was dependent on the activation of μOR, we investigated by immunogold cytochemistry the effects of intraplantar CFA injection on the plasma membrane density of δOR in μOR knockout (KO) mice. In untreated wild-type (WT) mice, only a small proportion of δOR was associated with neuronal plasma membranes in the dorsal horn of the spinal cord. The CFA-induced inflammation produced a significantly higher ratio of plasma membrane to intracellular receptors, as well as a 75% increase in the membrane density of immunoreactive δOR, in dendrites of the ipsilateral dorsal horn as compared to untreated mice. This increase in the membrane density of δOR was likely due to a recruitment of receptors from intracellular stores since no difference in the overall δOR immunolabeling density was evident between CFA-treated and untreated mice. Most importantly, the CFA-induced changes in δOR plasma membrane insertion seen in WT animals were not present in the spinal cord of μOR KO mice. These results demonstrate that the integrity of μOR is necessary for CFA-induced changes in δOR trafficking to occur and suggest that these changes could be elicited by stimulation of μOR by endogenous opioids released in response to chronic inflammatory pain.

Acute and subchronic MPTP administration differentially affects striatal glutamate synaptic function

We previously reported that 1 month following unilateral loss (>95%) of striatal dopamine, there is an increase in striatal glutamate function as measured by in vivo microdialysis and quantitative immuno-gold electron microscopy (Meshul, C.K., et al., 1999, Neuroscience 88, 1–16). The goal of this study was to determine the effect of bilateral loss of striatal dopamine on striatal glutamate function following acute or subchronic administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to C57/B6J mice. Animals were administered either single injections (ip) of 30 mg/kg/day for 7 days (subchronically treated group) or 20 mg/kg × 4 doses every 2 h (acutely treated group) of the toxin or saline. One month following the first injection, there was a 44 and 65% loss in the relative density of tyrosine hydroxylase (TH) immunolabeling within the dorsolateral striatum in the subchronically and acutely MPTP-treated groups compared to the saline group, respectively. There was a decrease in the basal level of extracellular glutamate within the striatum in the subchronically MPTP-treated animals compared to an increase in the acutely treated group in relationship to the saline group. Ultrastructurally, only in the acutely MPTP-treated group was there a decrease in the density of glutamate immunolabeling within nerve terminals associated with an asymmetrical synaptic contact in the dorsolateral striatum compared to either the subchronic or saline groups. In addition, there was a decrease in the relative density of GluR-2/3 subunit immunolabeling within the dorsolateral striatum in the acute MPTP compared to the saline group. These data indicate that differences in striatal glutamate function appear to be associated with the dosing interval of MPTP administration and the variable loss of striatal TH immunolabeling.

Nicotine Alters Striatal Glutamate Function and Decreases the Apomorphine-Induced Contralateral Rotations in 6-OHDA-Lesioned Rats

Theoverall goal of this study was to determine the effects of subchronic nicotine (0.4 mg/kg) treatment for 7 or 14 days on striatal glutamate function in both naı̈ve and in 6-hydroxydopamine (6-OHDA)-treated rats in which the nigrostriatal dopamine pathway was lesioned. In lesioned animals, the effect of nicotine on apomorphine-induced contralateral rotations was also assessed. In naı̈ve rats, once daily nicotine administration for 7 or 14 days resulted in a decrease and then an increase, respectively, in the basal extracellular level of striatal glutamate compared to the saline-treated group. Ultrastructurally, 14-day treatment with nicotine resulted in an increase in the density of striatal glutamate immunolabeling within nerve terminals making an asymmetrical synaptic contact compared to the saline-treated group. In 6-OHDA-lesioned animals, coadministration of nicotine with apomorphine or nicotine alone for 7 days resulted in an increase in the density of nerve terminal glutamate immunolabeling, compared to the apomorphine- or saline-treated groups. However, coadministration of nicotine with apomorphine for 14 days resulted in a decrease in the density of nerve terminal glutamate immunolabeling compared to the nicotine-treated group. Following subchronic treatment of 6-OHDA-lesioned rats with apomorphine for 7 or 14 days, there was an increase in the number of apomorphine-induced contralateral rotations compared to the saline treated group. There was a decrease in the number of apomorphine-induced contralateral rotations in the group coadministered nicotine with apomorphine for 7 or 14 days compared to the apomorphine treated group. The data suggests that in this 6-OHDA lesion model of Parkinson's disease, treatment with nicotine may be useful in counteracting the increased behavioral effect (i.e., contralateral rotations) observed after treatment with a dopamine agonist, such as apomorphine.

Expression of p75 NTR in photoreceptor cells of dystrophic rat retinas

Although a gene mutation in the Royal College of Surgeons (RCS) dystrophic rat results in defective phagocytosis and in accumulation of debris in the subretinal space, the molecular mechanisms leading to photoreceptor cell death remain unclear. In this study, the expression of p75 NTR, the low-affinity neurotrophin receptor incriminated in the apoptosis of developing neurons, was investigated at various stages of retinal degeneration in dystrophic rats using immunohistochemistry, in situ reverse transcription polymerase chain reaction (RT-PCR), Western blot, and relative RT-PCR. In normal adult retinas, p75 NTR immunolabeling was observed mainly in the outer limiting membrane, with punctate labeling in the inner nuclear and ganglion cell layers. In 18- to 30-day-old dystrophic retinas, the immunostaining appeared to increase especially in the photoreceptor outer and inner segments. Dense staining was also observed in the retinal pigment epithelium (RPE) and choroid. In 60-day-old dystrophic rat retinas, the density of immunolabeling for p75 NTR increased dramatically in the remaining inner retina, especially in the inner nuclear, inner plexiform, and ganglion cell layers. Post-embedding immunogold labeling of normal retinas verified the distribution of p75 NTR in photoreceptor cells within the inner segments, cell bodies, and outer segments. The apparent increased intensity in p75 NTR immunostaining in dystrophic retinas was verified by Western blots and densitometric analyses. In situ RT-PCR and relative RT-PCR further established increased synthesis of p75 NTR in dystrophic retinas. The increased levels of p75 NTR in the RPE and photoreceptor cells, the initial sites of injury, during retinal degeneration in dystrophic rats strongly suggest that altered expression of p75 NTR may be directly involved in photoreceptor death.