Quantitative autoradiography of [3H]forskolin binding sites in post-mortem brain staged for Alzheimer's disease neurofibrillary changes and amyloid deposits

Adenylyl cyclase (AC) signal transduction has been shown to be affected in Alzheimer's disease (AD). Deficits have been described in different components of the system, from the receptor to the effector level. [³H]forskolin is a diterpene that binds with high affinity to AC. In the present report, we used autoradiography to study [³H]forskolin binding to sections of entorhinal cortex and hippocampus from 23 cases staged for AD pathology according to Braak and Braak [Acta Neuropathol. 82 (1991) 239–259]. This protocol defines six stages according to neurofibrillary changes, which start in the entorhinal region (stages I–II), spread to the hippocampus (stages III–IV) and finally to the isocortical areas (stages V–VI). The amyloid classification includes three stages in which the basal isocortex is first affected (stage A), followed by other isocortical association areas (stage B) and finally the primary isocortical areas (stage C). We also studied the effects of the GTP-analogue Gpp[NH]p on binding, in order to detect changes in G-protein–AC coupling. We used two different concentrations of Gpp[NH]p, that were previously reported to inhibit and stimulate [³H]forskolin binding via G_i and G_s, respectively. Results showed that [³H]forskolin binding declined significantly with staging for neurofibrillary changes only in the entorhinal region (P<0.05, ANOVA). In addition, the decrease in [³H]forskolin binding observed in the presence of 1 μM Gpp[NH]p diminished significantly with staging in the entorhinal region (P<0.05, ANOVA). No significant changes were seen with amyloid staging, with the exception of the CA1 subfield of the hippocampus, where [³H]forskolin binding in the absence of Gpp[NH]p was significantly decreased at stage B compared with all other stages (P<0.05, ANOVA). In conclusion, our results showed a very limited decrease in [³H]forskolin binding with the progression of AD pathology, suggesting that the AC levels may be largely preserved in the disease. The specific change in the effect of a low concentration of Gpp[NH]p on the binding could indicate the loss of Ca²⁺/calmodulin-sensitive AC isoforms in AD.     

Interruptions of early cortical development affect limbic association areas and social behaviour in rats; possible relevance for neurodevelopmental disorders

Deficits in social behaviour are found in several neuropsychiatric disorders with a presumed developmental origin. Adequate social behaviour may rely importantly on the associative integration of new stimuli with previously stored, related information. The limbic allocortex, in particular the entorhinal region, is thought to support this kind of processing. Therefore, in the present study, gestating dams were treated with methylazoxymethanol acetate (MAM) on one of gestational days nine to twelve, to interrupt neuronal proliferation in the entorhinal region of the developing foetuses. Effects of prenatal MAM administration on social behaviour were evaluated in adult animals. As the entorhinal cortex has been implicated by some studies in spatial memory, effects on this function were also investigated. Following the behavioural studies, brain morphology was screened for effects of MAM. Our results show moderate to severe social impairment in MAM-treated animals, depending on the exact timing of prenatal exposure. By contrast, spatial reference and working memory were not importantly affected in any group. Analysis of brain morphology in the MAM-treated offspring supported maldevelopment of the entorhinal cortex and revealed mild abnormalities also in some connected limbic and limbic affiliated structures, such as the perirhinal and ectorhinal cortex, the anterior cingulate cortex and the medial septum-diagonal band region. Findings are discussed with respect to entorhinal cortex function, and with regard to their relevance for psychiatric disorders with a putatively neurodevelopmental pathogenesis, such as schizophrenia.     

Adenosine acting via A1 receptors, controls the transition to status epilepticus-like behaviour in an in vitro model of epilepsy

Adenosine has powerful inhibitory effects in the central nervous system. In this study, we aim to understand how adenosine controls the progression of seizure-like events (SLEs) in a seizure-prone region of the brain, the entorhinal cortex. We chose to use a low Mg(2+) model of epilepsy in an in vitro slice preparation where, in the entorhinal cortex, SLEs progress into a type of epileptiform activity called late recurrent discharges (LRDs) that bear resemblance to status epilepticus. Adenosine, acting via its A1 receptor, exerted powerful inhibitory effects to prevent the spontaneous progression to LRDs while the potent A1 receptor antagonist, DPCPX, accelerated the progression in a concentration dependent manner. The spontaneous progression from SLEs to LRDs was associated with a decline in total cellular ATP levels and studies with metabolic inhibitors indicated a key role for the production of endogenous adenosine from ATP. We therefore hypothesise that when ATP becomes rate limiting, extracellular adenosine levels fall, the normal inhibitory brake is removed and the progression from SLEs to LRDs or status epilepticus-like activity can ensue. Moreover, under these conditions, inhibition of the adenine nucleotide salvage pathways reversed the status epilepticus-like activity. Our findings suggest a powerful role for adenosine for the control of the progression to status epilepticus-like activity in an epilepsy model that is refractory to most anti-epileptic drugs. On this basis, manipulation of adenine nucleotide metabolism may represent a potential therapeutic approach for the treatment of status epilepticus.     

Environmental contaminant-mixture effects on CNS development, plasticity, and behavior

Environmental contaminants within the polycyclic aromatic hydrocarbon (PAH) and halogenated aromatic hydrocarbon class have been shown to cross the placenta exposing the fetus to the contaminant body burden of the mother. Consequently, a gestational exposure to environmental contaminants may result in increased adverse health outcomes, possibly affecting cognitive performance. Benzo(a)pyrene [B(a)P] and 2,3,7,8, tetrachlorodibenzo-p-dioxin (TCDD) are two prototypical environmental contaminants. A systematic review of the literature suggests that there may be a relationship between vulnerability in susceptible populations and health disparities. The purpose of this mini-review is to provide a point of reference for neurotoxicological studies of environmental contaminant mixture effects on indices of development in general, and on neurodevelopment in particular. Environmental contaminant-mixture-induced decrements in (1) birth index, (2) N-methyl-D-aspartate receptor (NMDA) mRNA expression, (3) long-term potentiation (LTP), (4) fixed-ratio performance learning behavior, and (5) experience-dependent activity related cytoskeletal-associated protein (Arc) mRNA and protein expression collectively support associations between neurobehavioral deficits and gestational exposure to environmental levels of these contaminants. Collectively, data are presented in this mini-review evaluating the effect of gestational exposure to environmental contaminant-mixtures on specific indices of learning and memory, including hippocampal-based synaptic plasticity mechanisms. These indices serve as templates for learning and memory, and as such, from a vulnerability perspective, may serve as targets for dysregulation during development in susceptible populations that have been disproportionately exposed to these contaminants. Included in this review is also a discussion of the relevance of developing biomarkers for use within the framework of cumulative risk-assessment.     

Impaired activation of CA3 pyramidal neurons in the epileptic hippocampus

We employed in vitro and ex vivo imaging tools to characterize the function of limbic neuron networks in pilocarpine-treated and age-matched, nonepileptic control (NEC) rats. Pilocarpinetreated animals represent an established model of mesial temporal lobe epilepsy. Intrinsic optical signal (IOS) analysis of hippocampal-entorhinal cortex (EC) slices obtained from epileptic rats 3 wk after pilocarpine-induced status epilepticus (SE) revealed hyperexcitability in many limbic areas, but not in CA3 and medial EC layer III. By visualizing immunopositivity for FosB/ΔFosBrelated proteins—which accumulate in the nuclei of neurons activated by seizures—we found that: (1) 24 h after SE, FosB/ΔFosB immunoreactivity was absent in medial EC layer III, but abundant in dentate gyrus, hippocampus proper (including CA3) and subiculum; (2) FosB/ΔFosB levels progressively diminished 3 and 7 d after SE, whereas remaining elevated (p<0.01) in subiculum; (3) FosB/ΔFosB levels sharply increased 2 wk after SE (and remained elevated up to 3 wk) in dentate gyrus and in most of the other areas but not in CA3. A conspicuous neuronal damage was noticed in medial EC layer III, whereas hippocampus was more preserved. IOS analysis of the stimulus-induced responses in slices 3 wk after SE demonstrated that IOSs in CA3 were lower (p<0.05) than in NEC slices following dentate gyrus stimulation, but not when stimuli were delivered in CA3. These findings indicate that CA3 networks are hypoactive in comparision with other epileptic limbic areas. We propose that this feature may affect the ability of hippocampal outputs to control epileptiform synchronization in EC.     

The extent of neurofibrillary pathology in perforant pathway neurons is the key determinant of dementia in the very old

Neurofibrillary pathology as found in Alzheimer’s disease (AD) is also found in the normal elderly, suggesting that these changes may be part of the aging process. In this study, we assessed the densities and distribution of structures recognized by the monoclonal antibody (mAb) to phosphorylated tau (AT8) in the hippocampal formation and medial temporal isocortex of 19 centenarians. Of these, 4 cases were demented and 15 non-demented. AT8 immunoreactivity correlated with the global deterioration scale (GDS). The density of both intraneuronal neurofibrillary tangles (I-NFTs) and neuritic clusters (NCs) significantly correlated with the GDS in the layer II of the entorhinal cortex (r = 0.66, P = 0.005 and r = 0.611, P = 0.01, respectively). Density of I-NFTs in the subiculum (r = 0.491; P = 0.034) also correlated significantly. No other area was found to be statistically significant. Importantly, no correlation was found when demented and non-demented centenarian cases were analyzed separately, suggesting that the difference marks a fundamental shift between AD and non-demented individuals. This assertion is supported by the significantly higher densities of I-NFTs and NCs in the transentorhinal (P = 0.043 and P = 0.011, respectively) and layer II of the entorhinal cortex (P = 0.02 and P = 0.007, respectively), and I-NFTs in the subiculum (P < 0.001) and CA1 (P = 0.011) in the demented group when compared with the non-demented cases. Granular diffuse deposits, an early stage parameter of the neurofibrillary pathology involving accumulation of non-fibrillar abnormally phosphorylated tau protein did not correlate with the GDS or between the two groups studied. This study, combining morphometric and confocal analyses, not only provides further evidence that, in the brains of patients with AD, the perforant pathway is highly sensitive to tau pathology but also that involvement is distinct from the changes of normal aging, even of the oldest old. Received: 23 April 1999 / Revised: 27 July 1999, 11 October 1999 / Accepted: 12 October 1999     

Excitatory and inhibitory control of epileptiform discharges in combined hippocampal/entorhinal cortical slices

We examined whether epileptiform activity can be induced and prevented by mild reduction of GABA_A receptor-mediated inhibition and non-NMDA receptor-mediated excitation, respectively, in different regions of combined hippocampal/entorhinal cortical slices from juvenile rats (P15–21). We used the receptor antagonists bicuculline (GABA_A) and CNQX (non-NMDA) as tools to investigate the sensitivities of the CA1, the subiculum (SUB) and the medial entorhinal cortex (MEC) for generating epileptiform discharges upon extracellular stimulation. We found that low concentrations of bicuculline (<3.5 μM) were enough to induce epileptiform discharges in the three regions. These discharges were similar to those observed under high concentrations of bicuculline (>10 μM) and consisted of stereotyped population bursts, recorded both extra- and intracellularly. Interestingly, the CA1 and SUB were more susceptible to generate discharges compared to the MEC in the same slices. We also found that non-NMDA excitation was critical in controlling discharges, as they were blocked by CNQX in a concentration-dependent manner. The sensitivity of the CA1 region to CNQX was lower than that of the SUB and MEC. Based on these regional differences, we show that epileptiform activity can be pharmacologically isolated within the CA1 region in the hippocampal–entorhinal circuitry in vitro.     

A subset of calretinin-positive neurons are abnormal in Alzheimer's disease

The distribution of the calcium-binding protein calretinin was investigated by immunohistochemistry in the hippocampus, the subicular areas, and the entorhinal cortex in patients with Alzheimer's disease and in control subjects. By double immunolabelling, the calretinin immunoreactivity was compared to the immunoreactivity for /A4 amyloid or for tau proteins. Calretinin-positive neurons were mainly observed in the molecular layer of the gyrus dentatus, the stratum radiatum of the Ammon's horn, and in layers II and III of the entorhinal cortex. The general pattern of calretinin immunoreactivity was conserved in Alzheimer's disease. Calretinin-positive neurons appeared normal in the hippocampus but had a reduced dendritic tree in the entorhinal cortex. Dystrophic calretinin immunoreactive fibres were often observed in the outer molecular layer of the gyrus dentatus and in the CA4 sector in Alzheimer's disease. Most neurons containing neurofibrillary tangles were not calretinin immunoreactive and most senile plaques were not associated with calretinin positive fibres. These results show that entorhinal calretinin-positive neurons are affected in Alzheimer's disease in spite of an absence of systematic association with neurofibrillary tangles and senile plaques.Supported by grants from the Belgian FRSM (3.4504.91, 3.4517.92), the Fonds de Recherche Divry, the Fondation M.T. de Lava and Alzheimer Belgique     

The effects of intrahippocampal ibotenate on resistance to extinction after continuous or partial reinforcement

Summary Intracerebral injections of ibotenate were used to produce, in rats, extensive cell loss in the hippocampus and dentate gyrus (complete hippocampal, CH), in the CH plus subiculum (SUB + CH), or in the subiculum plus entorhinal cortex (SUB + EC). These rats and sham-operated controls were trained to run in a straight alley for food reward delivered on a continuous (CR) or partial (PR) reinforcement schedule. In controls PR training gave rise to the well-known partial reinforcement extinction effect (PREE), i.e., greater resistance to extinction than that observed in CR-trained animals. Previous work had shown that large aspiration lesions of the hippocampal formation eliminate the PREE by increasing resistance to extinction in CR-trained animals and decreasing resistance to extinction in PR-trained animals. In the present experiments the PREE survived CH lesions, which increased resistance to extinction in both CR and PR training conditions; these effects were observed in the start and run (but not goal) sections of the alley. In contrast, subicular cell loss (in both SUB + CH and SUB + EC groups) abolished the PREE (but in the goal section only) by increasing resistance to extinction in the CR condition and decreasing resistance to extinction in the PR condition. In addition, some of the effects of PR training on start and run speeds during acquisition were altered by the CH and SUB + CH lesions. These results confirm previous data showing that the hippocampal formation plays a role in mediating the behavioural effects of PR training, but require modification of the model previously proposed to account for these data.     

An electrophysiological analysis of chronic ethanol neurotoxicity in the dentate gyrus: Distribution of entorhinal afferents

Summary This study investigated the persistent effects of chronic ethanol consumption on the distribution of entorhinal afferents to stratum moleculare of the dentate gyrus. Rats were maintained on ethanol, or sucrose-containing liquid diets, for a period of 20 weeks but were withdrawn from the special diet for at least 8 weeks prior to acute electrophysiologic recordings. One-dimensional laminar analyses were obtained by stepping a microelectrode in 25 m increments across both the dorsal and ventral blades of the dentate gyrus and sampling the field potentials at each point. Current-source density (CSD) was calculated from the field potential data.Electrical stimulation of the angular bundle elicited a short-latency, negative field potential covering the outer 2/3 of the molecular layer. CSD analysis revealed a major current sink in stratum moleculare bounded by a major current source, localized to the hilus, granule cell layer, and proximal stratum moleculare, and a minor current source localized to the outer molecular layer. Chronic ethanol treatment resulted in (1) a significant shrinkage of the spatial extent of the current sink in stratum moleculare, (2) a significant reduction in the distance from the peak inward synaptic current to the granule cell layer and (3) no change in the distance from the proximal inversion point to the granule cell layer. Taken together, these results indicate a loss of entorhinal afferents in the outer molecular layer. Coupled with available anatomical evidence, these results suggest that chronic ethanol treatment produces a preferential loss of lateral entorhinal afferents to the dentate gyrus.Supported by funds from the Medical Research Service of the Veterans Administration, NIAAA Grant AA-00200 to Don W. Walker, NIAAA predoctoral fellowship AA-05153 to WCA and NINCDS grant NS-10229 to William Brownell