Colocalization of mu-opioid receptors and activated G-proteins in rat cingulate cortex.

Anterior cingulate cortex (ACC) has a role in pain processing, however, little is known about opioid system organization and actions. This rodent study defines opioid architecture in the perigenual and midcingulate divisions of ACC, relates mu-opioid receptor binding and G-protein activation, and localizes such binding to afferent axons with knife-cut lesions and specifically to noradrenergic terminals with immunotoxin lesions (anti-dopamine beta-hydroxylase-saporin; anti-DBH-saporin). [(3)H]Tyr-D-AlaGly-MePhe-Gly-ol (DAMGO) binding was highest in perigenual areas 32 and 24 with a peak in layer I. Midcingulate area 24' and posterior cingulate area 29 had overall lower binding in each layer. In contrast, DAMGO-stimulated [(35)S]guanosine-5'-O-(gamma-thio)-triphosphate (GTPgammaS) binding in area 24' was similar to that in area 24, whereas area 29 had low and homogeneous binding. Undercut lesions reduced [(3)H]DAMGO binding in all layers with the greatest loss in layer I (-65%), whereas DAMGO-stimulated [(35)S]GTPgammaS binding losses occurred in only layers I-III. Anti-DBH-saporin reduced [(3)H]DAMGO binding in layer I of area 24; DAMGO-stimulated [(35)S]GTPgammaS binding was unchanged in areas 24' and 29. Correlation analysis of receptor and G-protein activation before and after undercut lesions suggested there were a greater number of DAMGO receptor sites for each G-protein on axons, than on somata and proximal dendrites. Finally, perigenual and midcingulate cortices have different opioid architectures due to a higher proportion of mu-opioid receptors expressed by afferent axons in areas 24 and 32.

Cytology of human caudomedial cingulate, retrosplenial, and caudal parahippocampal cortices.

Brodmann showed areas 26, 29, 30, 23, and 31 on the human posterior cingulate gyrus without marking sulcal areas. Histologic studies of retrosplenial areas 29 and 30 identify them on the ventral bank of the cingulate gyrus (CGv), whereas standardized atlases show area 30 on the surface of the caudomedial region. This study evaluates all areas on the CGv and caudomedial region with rigorous cytologic criteria in coronal and oblique sections Nissl stained or immunoreacted for neuron-specific nuclear binding protein and nonphosphorylated neurofilament proteins (NFP-ir). Ectosplenial area 26 has a granular layer with few large pyramidal neurons below. Lateral area 29 (29l) has a dense granular layer II-IV and undifferentiated layers V and VI. Medial area 29 (29m) has a layer III of medium and NFP-ir pyramids and a layer IV with some large, NFP-ir pyramidal neurons that distinguish it from areas 29l, 30, and 27. Although area 29m is primarily on the CGv, a terminal branch can extend onto the caudomedial lobule. Area 30 is dysgranular with a variable thickness layer IV that is interrupted by large NFP-ir neurons in layers IIIc and Va. Although area 30 does not appear on the surface of the caudomedial lobule, a terminal branch can form less that 1% of this gyrus. Area 23a is isocortex with a clear layer IV and large, NFP-ir neurons in layers IIIc and Va. Area 23b is similar to area 23a but with a thicker layer IV, more large neurons in layer Va, and a higher density of NFP-ir neurons in layer III. The caudomedial gyral surface is composed of areas 23a and 23b and a caudal extension of area 31. Although posterior area 27 and the parasubiculum are similar to rostral levels, posterior area 36' differs from rostral area 36. Subregional flat maps show that retrosplenial cortex is on the CGv, most of the surface of caudomedial cortex is areas 23a, 23b, and 31, and the retrosplenial/parahippocampal border is at the ventral edge of the splenium. Thus, Brodmann's map understates the rostral extent of retrosplenial cortex, overstates its caudoventral extent, and abridges the caudomedial extent of area 23.

Chronic heroin self-administration desensitizes mu opioid receptor-activated G-proteins in specific regions of rat brain.

In previous studies from our laboratory, chronic noncontingent morphine administration decreased mu opioid receptor-activated G-proteins in specific brainstem nuclei. In the present study, mu opioid receptor binding and receptor-activated G-proteins were examined after chronic heroin self-administration. Rats were trained to self-administer intravenous heroin for up to 39 d, achieving heroin intake up to 366 mg. kg(-1). d(-1). mu opioid-stimulated [(35)S]GTPgammaS and [(3)H]naloxone autoradiography were performed in adjacent brain sections. Agonist-stimulated [(35)S]GTPgammaS autoradiography also examined other G-protein-coupled receptors, including delta opioid, ORL-1, GABA(B), adenosine A(1), cannabinoid, and 5-HT(1A). In brains from heroin self-administering rats, decreased mu opioid-stimulated [(35)S]GTPgammaS binding was observed in periaqueductal gray, locus coeruleus, lateral parabrachial nucleus, and commissural nucleus tractus solitarius, as previously observed in chronic morphine-treated animals. In addition, decreased mu opioid-stimulated [(35)S]GTPgammaS binding was found in thalamus and amygdala after heroin self-administration. Despite this decrease in mu-activated G-proteins, [(3)H]naloxone binding demonstrated increased mu opioid receptor binding in several brain regions after heroin self-administration, and there was a significant decrease in mu receptor G-protein efficiency as expressed as a ratio between agonist-activated G-proteins and mu receptor binding. No effects on agonist-stimulated [(35)S]GTPgammaS binding were found for any other receptor examined. The effect of chronic heroin self-administration to decrease mu-stimulated [(35)S]GTPgammaS binding varied between regions and was highest in brainstem and lowest in the cortex and striatum. These results not only provide potential neuronal mechanisms that may contribute to opioid tolerance and dependence, but also may explain why various chronic effects of opioids develop to different degrees.

Region-specific changes in 5-HT(1A) receptor-activated G-proteins in rat brain following chronic buspirone.

5-Hydroxytryptamine(1A) (5-HT(1A)) receptors, which activate inhibitory G-proteins, are implicated in psychiatric disorders including anxiety and depression. Studies suggest that chronic 5-HT(1A) receptor agonist administration alters 5-HT(1A) receptor function, but the effect of chronic treatment on 5-HT(1A) receptor-activated G-proteins is unclear. In this study, agonist-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate (GTPgammaS) binding was examined following chronic administration of buspirone. Brains were processed for [35S]GTPgammaS autoradiography using R(+)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) for 5-HT(1A) receptors or baclofen for GABA(B) receptors. Net 8-OH-DPAT-stimulated [35S]GTPgammaS binding was decreased by 25-30% in the septum and dorsal raphe nucleus of buspirone-treated animals. No significant changes in 8-OH-DPAT-stimulated [35S]GTPgammaS binding were found in the prefrontal, entorhinal or cingulate cortices or hippocampus in buspirone-treated rats. GABA(B) receptor-stimulated [35S]GTPgammaS binding was increased by 25% in the hippocampus, with no significant changes in any other region examined. These results demonstrate region-specific alterations in 5-HT(1A) and GABA(B) receptor-activated G-proteins following chronic buspirone treatment, which may contribute to the clinical effects of this drug.

Chronic delta9-tetrahydrocannabinol treatment produces a time-dependent loss of cannabinoid receptors and cannabinoid receptor-activated G proteins in rat brain.

Chronic treatment of rats with delta9-tetrahydrocannabinol (delta9-THC) results in tolerance to its acute behavioral effects. In a previous study, 21-day delta9-THC treatment in rats decreased cannabinoid activation of G proteins in brain, as measured by in vitro autoradiography of guanosine-5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) binding. The present study investigated the time course of changes in cannabinoid-stimulated [35S]GTPgammaS binding and cannabinoid receptor binding in both brain sections and membranes, following daily delta9-THC treatments for 3, 7, 14, and 21 days. Autoradiographic results showed time-dependent decreases in WIN 55212-2-stimulated [35S]GTPgammaS and [3H]WIN 55212-2 binding in cerebellum, hippocampus, caudate-putamen, and globus pallidus, with regional differences in the rate and magnitude of down-regulation and desensitization. Membrane binding assays in these regions showed qualitatively similar decreases in WIN 55212-2-stimulated [35S]GTPgammaS binding and cannabinoid receptor binding (using [3H]SR141716A), and demonstrated that decreases in ligand binding were due to decreases in maximal binding values, and not ligand affinities. These results demonstrated that chronic exposure to delta9-THC produced time-dependent and region-specific down-regulation and desensitization of brain cannabinoid receptors, which may represent underlying biochemical mechanisms of tolerance to cannabinoids.

Multivariate analysis of laminar patterns of neurodegeneration in posterior cingulate cortex in Alzheimer's disease.

Posterior cingulate cortex is the site of earliest reductions in glucose metabolism and qualitatively different laminar patterns of neurodegeneration in Alzheimer's disease (AD). This study used multivariate analyses of area 23 in 72 cases of definite AD to assess relationships between laminar patterns of neurodegeneration, neurofibrillary tangle (NFT) and senile plaque (SP) densities, age of disease onset and duration, and apolipoprotein E (ApoE) genotype. No age-related changes in neurons occurred over four decades in 17 controls and regression analysis of all AD cases showed no relationships between neuron, SP, and tau-immunoreactive NFT densities. Principal components analysis of neurons in layers III-Va and eigenvector projections showed five subgroups. The subgroups were independent because each had a full range of disease durations and qualitatively different laminar patterns in degeneration suggested disease subtypes (ST). Cases with most severe neuron losses (STSevere) had an early onset, most SP, and highest proportion of ApoE epsilon4 homozygotes. Changes in the distribution of NFT were similar over disease course in two subtypes and NFT did not account for most neurodegeneration. In STII-V with moderate neuron loss in most layers, cases with no NFT had a disease duration of 3.5 +/- 0.9 years (mean +/- SEM), those with most in layers IIIc or Va had a duration of 7.3 +/- 1 years, and those with most in layers II-IIIab had a duration of 12.1 +/- 1 years. In STSevere, cases with highest NFT densities in layers II-IIIab also were late stage. Finally, epsilon4 homozygotes were most frequent in STSevere, but four statistical tests showed that this risk is not directly involved in neurodegeneration. In conclusion, multivariate pattern recognition shows that AD is composed of independent neuropathological subtypes and NFT in area 23 do not account for most neuron losses.

PCR-based apolipoprotein E genotype analysis from archival fixed brain.

A technique is described for determining the apolipoprotein E genotype (apo E; alleles epsilon2, epsilon3, or epsilon4) from tissues which have been fixed with 4-10% formaldehyde and archived. The procedure requires efficient extraction and exhaustive purification of DNA from the fixed tissue. Because the fixation process renders the DNA largely crosslinked and/or sheared (therefore unsuitable for traditional analysis), a nested polymerase chain reaction (PCR) is employed (using two apo E gene specific primer pairs) to specifically amplify the polymorphic region of the gene. The genotype was then determined using previously reported HhaI polymorphisms that occur as a direct result of the variant codons responsible for the three alleles. This protocol permitted the successful genotyping of 90% (34 out of 38) of the archived brain samples from Alzheimer's disease (AD) patients. These samples included such extremes as a sample that had been stored for 12 years in formalin. This procedure permits the retrospective analysis of samples that had been processed and stored well before the original characterization of apo E alleles as risk factors in AD. Finally, this approach is readily adapted to the analysis of any gene of interest, whether by restriction fragment length polymorphism or direct amplicon DNA sequencing. It is also a very robust assay for less stringent conditions such as DNA isolated from whole blood or frozen tissue.

Human cingulate cortex: surface features, flat maps, and cytoarchitecture.

The surface morphology and cytoarchitecture of human cingulate cortex was evaluated in the brains of 27 neurologically intact individuals. Variations in surface features included a single cingulate sulcus (CS) with or without segmentation or double parallel sulci with or without segmentation. The single CS was deeper (9.7 +/- 0.81 mm) than in cases with double parallel sulci (7.5 +/- 0.48 mm). There were dimples parallel to the CS in anterior cingulate cortex (ACC) and anastomoses between the CS and the superior CS. Flat maps of the medial cortical surface were made in a two-stage reconstruction process and used to plot areas. The ACC is agranular and has a prominent layer V. Areas 33 and 25 have poor laminar differentiation, and there are three parts of area 24: area 24a adjacent to area 33 and partially within the callosal sulcus has homogeneous layers II and III, area 24b on the gyral surface has the most prominent layer Va of any cingulate area and distinct layers IIIa-b and IIIc, and area 24c in the ventral bank of the CS has thin layers II-III and no differentiation of layer V. There are four caudal divisions of area 24. Areas 24a' and 24b' have a thinner layer Va and layer III is thicker and less dense than in areas 24a and 24b. Area 24c' is caudal to area 24c and has densely packed, large pyramids throughout layer V. Area 24c' g is caudal to area 24c' and has the largest layer Vb pyramidal neurons in cingulate cortex. Area 32 is a cingulofrontal transition cortex with large layer IIIc pyramidal neurons and a dysgranular layer IV. Area 32' is caudal to area 32 and has an indistinct layer IV, larger layer IIIc pyramids, and fewer neurons in layer Va. Posterior cingulate cortex has medial and lateral parts of area 29, a dysgranular area 30, and three divisions of area 23: area 23a has a thin layer IIIc and moderate-sized pyramids in layer Va, area 23b has large and prominent pyramids in layers IIIc and Va, and area 23c has the thinnest layers V and VI in cingulate cortex. Area 31 is the cinguloparietal transition area in the parasplenial lobules and has very large layer IIIc pyramids. Finally, variations in architecture between cases were assessed in neuron perikarya counts in area 23a. There was an age-related decrease in neuron density in layer IV (r = -0.63; ages 45-102), but not in other layers.(ABSTRACT TRUNCATED AT 400 WORDS)

AF-DX 384 binding in rabbit cingulate cortex: two site kinetics and section autoradiography.

Autoradiographic studies of muscarinic receptors are limited by the lack of selective ligands. Inasmuch as AF-DX 384 has a higher affinity for m2 than m4 receptors and pirenzepine (PZ) has a reverse affinity profile, competition between these ligands was used to label m2 receptors in homogenized and sectioned tissue. Rabbit cingulate cortex was used because m2 receptors are expressed by anterior thalamic axons in posterior cingulate cortex (PCC) and this region is easily deafferented with undercut lesions to demonstrate presynaptic binding. Saturation isotherms and Scatchard analysis of [3H]AF-DX 384 binding showed one binding site with a KD of 9 +/- 2.3 nM (mean +/- SEM) and a Bmax of 1405 +/- 146 fmol/mg protein. Competition studies with [3H]AF-DX 384 (2 nM) and 10(-10)-10(-4) M PZ were performed in anterior cingulate cortex (ACC) and PCC. In both regions, the best fit was a two site model for low (BL) and high (BH) affinity binding in which Bmax values were similar (ACC: BL = 535 +/- 62 fmol/mg, BH = 676 +/- 85; PCC: BL = 552 +/- 41; BH = 675 +/- 85). Although affinities for KH were similar in each region (ACC: KH = 4.69 +/- 1.36 nM; PCC: KH = 8.53 +/- 3.69 nM), those for KL were significantly different (ACC: 181 +/- 15.4 nM; PCC: 285 +/- 42; P = .018). Binding of [3H]AF-DX 384 with PZ (150 nM) was best fit with a single site model (KD = 6 +/- 0.01 nM; Bmax = 688 +/- 31 fmol/mg), suggesting that PZ blocks the lower affinity site. (ABSTRACT TRUNCATED AT 250 WORDS)

Reorganization of cingulate cortex in Alzheimer's disease: neuron loss, neuritic plaques, and muscarinic receptor binding.

Pathology related to dementia of the Alzheimer type (DAT) develops later in cingulate cortex than in medial temporal areas. Therefore, end-stage cases have earlier forms of pathology in cingulate cortex, and postmortem studies of this region may provide a window on processes that temporal cortices pass through decades before death. Five classes of DAT have been described on the basis of neuron degeneration and receptor binding in posterior cingulate cortex. The present study assessed binding of 3H-oxotremorine-M with pirenzepine (OXO-M/PZ), a protocol for presynaptic muscarinic receptors, and thioflavin S-stained neuritic plaques (NPs) in cingulate area 23a in 12 DAT cases distributed over four classes of pathology and in nine age-matched control cases. OXO-M/PZ binding was significantly elevated in layers I, II, IV, and VI of all DAT cases and was very high in layer V compared to control cases. Almost 75% of the layer Va increase was due to binding in classes 2 and 3, while classes 1 and 4 were least affected. In class 3 cases, neuron density in layer Va was inversely correlated with OXO-M/PZ binding (r = -0.98) and primitive NP densities (r = -0.93). The close association between neuron densities and presynaptic muscarinic ligand binding in some classes confirms that there are independent classes of DAT. The high and inverse correlations between cortical pathology and ligand binding in class 3 cases suggest that there is a progression in class 3 pathology. Finally, elevated OXO-M/PZ binding and a report of increased choline uptake suggest that cholinergic axons sprout in DAT, and this sprouting may be associated with a progressive loss of postsynaptic elements.

Limbic thalamus in rabbit: architecture, projections to cingulate cortex and distribution of muscarinic acetylcholine, GABAA, and opioid receptors.

Nuclei of the thalamus that project to cingulate cortex have been implicated in responses to noxious stimuli, cholinergic and motor functions. The rabbit limbic thalamus may play an important role in these functions, but has not been studied extensively in terms of its cytoarchitecture, the topographical organization of its cortical projections, and differential transmitter regulation of its subnuclei. Therefore, the architecture, projections to cingulate cortex, and radioligand binding were investigated in the anterior, ventral, lateral, and midline nuclei of rabbit thalamus. The anterior nuclei are highly differentiated because both the dorsal and ventral nuclei have parvicellular and magnocellular divisions. Fluorescent dyes were injected into cingulate cortex to evaluate limbic thalamocortical connections. The anterior medial, submedial, and parafascicular nuclei project primarily to anterior cingulate cortex, while they have small or no projections to posterior areas. The ventral anterior and ventral lateral nuclei have a significant projection to dorsal cingulate cortex, including areas 24b and 29d. Projections of the anterior ventral nucleus are topographically organized, since medial parts of the parvicellular division project to rostral area 29, and lateral parts project to caudal area 29. The lateral nuclei and the parvicellular and magnocellular divisions of the anterior dorsal nucleus project with progressively higher densities in the rostrocaudal plane of area 29. Finally, the magnocellular division of the anterior ventral nucleus projects almost exclusively to caudal and ventral area 29, i.e., granular retrosplenial cortex. Ligand binding studies employed coverslip autoradiography and single grain counting techniques. Muscarinic receptor binding was moderate for both pirenzepine and oxotremorine-M in the parvicellular anterior ventral nucleus, while in other nuclei, there was an inverse relationship in the binding for these ligands. Most notably, the anterior dorsal nucleus, which receives no cholinergic input, had very high oxotremorine-M and low pirenzepine binding, while the anterior medial nucleus, which receives a moderate cholinergic input, had the highest pirenzepine binding and very low oxotremorine-M binding. Muscimol binding to GABAA receptors was highest in the anterior ventral nucleus, while it was at moderate levels in the anterior dorsal and lateral nuclei. The binding of Tyr-D-Ala-Gly-MePhe-Gly-ol to mu opioid receptors and 2-D-penicillamine-5-D-penicillamine-enkephalin to delta opioid receptors were both high in the parvicellular and low in the magnocellular divisions of the anterior dorsal nucleus. The magnocellular division of the anterior ventral, the lateral dorsal, and the parafascicular nuclei had high mu opioid binding, while the lateral dorsal and lateral magnocellular nuclei had low levels of delta opioid binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Muscarinic receptor binding increases in anterior thalamus and cingulate cortex during discriminative avoidance learning.

Training-induced neuronal activity develops in the mammalian limbic system during discriminative avoidance conditioning. This study explores behaviorally relevant changes in muscarinic ACh receptor binding in 52 rabbits that were trained to one of five stages of conditioned response acquisition. Sixteen naive and 10 animals yoked to criterion performance served as control cases. Upon reaching a particular stage of training, the brains were removed and autoradiographically assayed for 3H-oxotremorine-M binding with 50 nM pirenzepine (OXO-M/PZ) or for 3H-pirenzepine binding in nine limbic thalamic nuclei and cingulate cortex. Specific OXO-M/PZ binding increased in the parvocellular division of the anterodorsal nucleus early in training when the animals were first exposed to pairing of the conditional and unconditional stimuli. Elevated binding in this nucleus was maintained throughout subsequent training. In the parvocellular division of the anteroventral nucleus (AVp), OXO-M/PZ binding progressively increased throughout training, reached a peak at the criterion stage of performance, and returned to control values during extinction sessions. Peak OXO-M/PZ binding in AVp was significantly elevated over that for cases yoked to criterion performance. In the magnocellular division of the anteroventral nucleus (AVm), OXO-M/PZ binding was elevated only during criterion performance of the task, and it was unaltered in any other limbic thalamic nuclei. Specific OXO-M/PZ binding was also elevated in most layers in rostral area 29c when subjects first performed a significant behavioral discrimination. Training-induced alterations in OXO-M/PZ binding in AVp and layer Ia of area 29c were similar and highly correlated.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathological alterations in the amygdala in Alzheimer's disease.

The amygdala is severely and consistently affected by pathology in Alzheimer's disease. The distribution of Thioflavin S-stained neurofibrillary tangles and neuritic plaques was examined in the various nuclei that form the amygdala in 20 cases of clinically diagnosed Alzheimer's disease and five non-demented control cases. Large numbers of neurofibrillary tangles and neuritic plaques were observed in the accessory basal and cortical nuclei and the cortical transition area, while there was lesser involvement of the mediobasal nucleus. The medial, lateral, laterobasal and central nuclei were relatively spared. The distribution of neurofibrillary tangles and neuritic plaques was compared with neuroanatomic connections known from non-human primate experimental studies. This comparison suggests that (1) nuclei receiving and giving rise to hippocampal projections are consistently affected by neuropathological alterations in Alzheimer's disease; (2) the nuclei which receive strong cholinergic projections from the nucleus basalis of Meynert (e.g. laterobasal nucleus) have, in general, relatively few neuritic plaques; and (3) nuclei which receive olfactory projections are not uniformly affected, the cortical nucleus being heavily affected but the medial nucleus consistently spared.

Laminar distribution of neuron degeneration in posterior cingulate cortex in Alzheimer's disease.

The laminar distribution of neuron losses in posterior cingulate cortex were evaluated in 25 clinically and neuropathologically diagnosed cases of dementia of the Alzheimer type (DAT). The layer of maximal neuron loss in area 23a for each DAT case was determined by comparison with mean neuron densities for each layer of 17 neurologically intact control cases. The DAT cases were separated into five classes: class 1, 12% of all DAT cases, no or less than 40% neuron loss in any layer; class 2, 24%, maximal neuron losses in layers II or III; class 3, 28%, losses mainly in layer IV; class 4, 12%, losses mainly in layers V or VI; class 5, 24%, severe losses in all layers. An analysis of large and small neurons showed that in class 2 there was an equal loss of both in layer IIIa--b, in class 3 mostly small neurons were lost in layer IV, in class 4 mostly large neurons were lost in layers III, IV and V, while in class 5 there was no selectivity. The age of disease onset and length of the disease were the same for all classes, although classes 4 and 5 tended to have an earlier onset. No measures of thioflavin S-stained neuritic plaque (NP) or neurofibrillary tangle (NFT) density discriminated among these classes. In 64% of all DAT cases there was a progressive shift in NFT from ventral area 30 where most were in layer II to areas 23a--b where there was a balance between those in superficial and deep layers to dorsal area 23c where most were in layers V and VI.(ABSTRACT TRUNCATED AT 250 WORDS)