Zaineb Henderson

Aluminium toxicity in the rat brain: histochemical and immunocytochemical evidence

Although the neurotoxic actions of aluminium (Al) have been well documented, its contribution to neurodegenerative diseases such as Alzheimers disease remains controversial. In the present study, we applied histochemical techniques to identify changes induced by intracerebroventricular Al injections (5.4 microg in 5.5 microl, daily over a period of 5 successive days) in the adult rat brain after survival periods of either 1 or 6 weeks. For both Al- and saline-infused controls, no major signs of gross histological changes were evident in cresyl violet-stained sections. Al (as indicated by the fluorescent Morin staining) was concentrated in white matter of the medial striatum, corpus callosum, and cingulate bundle. Immunoreactivity of astrocytes and phagocytic microglia based on glial fibrillary acidic protein and ED1 markers, respectively, revealed a greater inflammatory response in Al-injected animals compared to controls. Damage of the cingulate bundle in Al-treated animals led to a severe anterograde degeneration of cholinergic terminals in cortex and hippocampus, as indicated by acetylcholinesterase labelling. Our data suggest that the enhancement of inflammation and the interference with cholinergic projections may be the modes of action through which Al may cause learning and memory deficits, and contribute to pathological processes in Alzheimers disease.

Differential effects of unaggregated and aggregated amyloid β protein (1–40) on K+ channel currents in primary cultures of rat cerebellar granule and cortical neurones

The effects of amyloid β protein on voltage‐gated K+ channel currents were studied using the whole‐cell patch‐clamp technique. The 1–40 amino acid form of amyloid β protein was applied to primary cultures of rat cerebellar granule and cortical neurones for 24 h. Both the unaggregated and aggregated forms of the peptide, which have differing biological activities, were used. In cerebellar granule neurones, 24‐h pre‐incubation with 1 µm unaggregated amyloid β protein resulted in a 60% increase in the ‘A’‐type component of K+ current. Increased delayed rectifier activity was Cd2+‐sensitive and was presumed to be secondary to an increase in voltage‐gated Ca2+ channel current activity. Unaggregated amyloid β protein had no effect on any component of the K+ channel current in cortical neurones. One micromolar of aggregated amyloid β protein had no effect on K+ channel current in either cell type but reduced cell survival within 24 h as measured using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL) assays. The unaggregated form of amyloid β protein had no neurotoxic effects when applied to either neurone type for up to 72 h. These data indicate that the unaggregated, non‐pathological form of amyloid β protein causes changes in the ion channel function of neurones, possibly reflecting a physiological role for the peptide.

Perineuronal nets ensheath fast spiking, parvalbumin‐immunoreactive neurons in the medial septum/diagonal band complex

Perineuronal nets, composed of extracellular matrix material, have previously been associated with parvalbumin‐immunoreactive neurons in the medial septum/diagonal band (MS/DB) complex of the rat. The aim of this study was to correlate the presence of perineuronal nets with electrophysiological properties and parvalbumin immunoreactivity in MS/DB neurons. Intracellular recordings were made from cells in a brain slice preparation maintained in vitro, and neurons were characterized into four populations: (i) slow‐firing neurons, (ii) burst‐firing neurons, (iii) fast spiking neurons with narrow action potentials and a small degree of spike frequency adaptation, and (iv) regular spiking neurons with broader action potentials and a high degree of spike frequency adaptation. Following electrophysiological characterization, neurons were filled with biocytin, processed for parvalbumin immunoreactivity and stained for perineuronal nets using Wisteria floribunda lectin. The three substances were viewed with triple fluorescence. Fast spiking, nonadapting neurons, shown previously to contain parvalbumin immunoreactivity, were nearly all ensheathed by perineuronal nets. There was a population of small parvalbumin‐immunoreactive neurons which did not possess perineuronal nets, and which were not encountered with the intracellular electrodes. The other three neuron types in the MS/DB did not contain parvalbumin immunoreactivity or perineuronal nets. In keeping with this neurochemical profile for electrophysiologically identified neurons, burst‐firing neurons had action potential parameters more similar to those of regular spiking than of fast spiking neurons. We conclude that fast spiking neurons, presumed to be GABAergic septohippocampal projection neurons, are surrounded by supportive structures to enable the high level of neuronal discharge required for producing disinhibition of hippocampal pyramidal neurons.

Hypoxic Depolarization of Cerebellar Granule Neurons by Specific Inhibition of TASK-1

Background and Purpose— The mechanisms underlying neuronal excitotoxicity during hypoxic/ischemic episodes are not fully understood. One feature of such insults is a rapid and transient depolarization of central neurons. TASK-1, an open rectifying K+ leak channel, is significant in setting the resting membrane potential of rat cerebellar granule neurons by mediating a standing outward K+ current. In this study we investigate the theory that the transient neuronal depolarization seen during hypoxia is due to the inhibition of TASK-1. Methods— Activity of TASK-1 in primary cultures of rat cerebellar granule neurons was investigated by the whole-cell patch-clamp technique. Discriminating pharmacological and electrophysiological maneuvers were used to isolate the specific channel types underlying acute hypoxic depolarizations. Results— Exposure of cells to acute hypoxia resulted in a reversible and highly reproducible mean membrane depolarization of 14.2±2.6 mV (n=5;P <0.01). Two recognized means of inhibiting TASK-1 (decreasing extracellular pH to 6.4 or exposure to the TASK-1–selective inhibitor anandamide) abolished both the hypoxic depolarization and the hypoxic depression of a standing outward current, identifying TASK-1 as the channel mediating this effect. Conclusions— Our data provide compelling evidence that hypoxia depolarizes central neurons by specific inhibition of TASK-1. Since this hypoxic depolarization may be an early, contributory factor in the response of central neurons to hypoxic/ischemic episodes, TASK-1 may provide a potential therapeutic target in the treatment of stroke.

Modulation of Ca2+ channel currents in primary cultures of rat cortical neurones by amyloid β protein (1-40) is dependent on solubility status

The Alzheimers disease peptide amyloid beta protein (Abeta) can exist in soluble and fibrillar, aggregated forms. Abeta in the aggregated form is thought to be pro-apoptotic, causing cell death when applied to cultured neurones by disrupting Ca(2+) homeostasis. This process may involve changes in Ca(2+) influx across the plasma membrane. The aim of this study was to quantify this effect by applying both the aggregated and unaggregated forms of Abeta to cultured rat cortical neurones. Unaggregated Abeta(1-40) (24-h pretreatment, 1 microM) stimulated an increase in voltage-dependent Ca(2+) channel current activity, which was found to comprise of N- and P-type current. In the aggregated form, Abeta(1-40) pre-treatment reduced Ca(2+) channel current density in cortical neurones via an action on N-type Ca(2+) current. This failure of aggregated Abeta(1-40) to increase the Ca(2+) channel current was confirmed on cerebellar granule neurone Ca(2+) currents which normally undergo an increase in activity following soluble Abeta application. Using the MTT and TUNEL assays, aggregated Abeta(1-40) was found to promote apoptotic cell death in cortical neurones confirming that Abeta exhibited the expected biological activity. Unaggregated Abeta had no neurotoxic effect. These data indicate that the unaggregated, non-pathological form of Abeta(1-40), and not the aggregated form, cause changes in neuronal Ca(2+) channel activity. This may reflect a normal functional role for amyloid peptides in the central nervous system.

Further evidence for the absence of a descending cholinergic projection from the brainstem to the spinal cord in the rat

Serotonergic and catecholaminergic neurons are known to project from the brainstem to the spinal cord. However, evidence for a bulbo-spinal projection that is cholinergic is sparse despite immunocytochemical and physiological evidence for a cholinergic influence on the cord. In this study we examined the possibility of a direct cholinergic bulbo-spinal projection in the rat using a combination of retrograde axonal tracing techniques and choline acetyltransferase immunocytochemistry. Although many cells were found to project to the cord from the brainstem, none were identified as being cholinergic, confirming previous evidence that the cholinergic innervation of the cord is intrinsic.

RESPONSES OF BASAL FOREBRAIN CHOLINERGIC NEURONS TO DAMAGE IN THE ADULT BRAIN

The basal forebrain cholinergic system, which projects to all cortical areas, is a good model for the study of the responses of central nervous system (CNS) neurons to injury. Much is known about the specific neurotrophic factors of basal forebrain cholinergic neurons, and there are many techniques available to chart the progress of degeneration and recovery of cholinergic neurons after damage. There is also a clinical version of damage to cholinergic neurons which is part of the selective pathology of Alzheimers disease. In general, CNS neurons do not regenerate well after brain damage. The first part of the review describes how the CNS promotes only limited neuronal regeneration, and that this is because of the presence of inhibitory factors and the lack of growth factors. Despite this, some CNS areas may provide a better environment for CNS regeneration than others. In the second part of the review, the degeneration and regeneration of basal forebrain cholinergic pathways in the adult rat are discussed. It is shown how the collateral sprouting of cholinergic axons occur more readily in the hippocampal formation than in neocortical areas, and that this could be linked with the more neuroplastic properties of the hippocampus. The third part of the review describes how the degeneration of cholinergic pathways in Alzheimers disease is likely to be secondary to the pathology or degeneration of certain cortical areas. The hypothesis is put forward that the severity of the pathology in highly plastic limbic cortical areas could be linked with their susceptibility to risk factors of Alzheimers disease such as ageing, and genetic and environmental factors.

Morphology of local axon collaterals of electrophysiologically characterised neurons in the rat medial septal/ diagonal band complex.

Neurons in the medial septal/diagonal band complex (MS/DB) in vivo exhibit rhythmic burst‐firing activity that is phase‐locked with the hippocampal theta rhythm. The aim was to assess the morphology of local axon collaterals of electrophysiologically identified MS/DB neurons using intracellular recording and biocytin injection in vitro. Cells were classified according to previous criteria into slow‐firing, fast‐spiking, regular‐spiking, and burst‐firing neurons; previous work has suggested that the slow‐firing neurons are cholinergic and that the other types are GABAergic. A novel finding was the existence of two types of burst‐firing neuron. Type I burst‐firing neurons had significantly longer duration after hyperpolarisation potentials when held at −60 mV, and at −75 mV, type I neurons exhibited a low‐threshold spike with more rapid activation and inactivation kinetics than those of type II neurons. We have, also for the first time, described the main features of the local axon collaterals of the five neuron types. All filled neurons possessed a main axon that gave forth 1–12 local primary axon collaterals. All electrophysiological types, except for the type I burst‐firing neuron, had a main axon that coursed toward the fornix. Myelination of the main axon was a prominent feature of all but the slow‐firing neurons. Branching of the primary axon collaterals of the fast‐spiking and type I burst‐firing neurons was more extensive than that of the other cell types, with those of the slow‐firing neurons exhibiting the least branching. All cell types possessed axon collaterals of the en passant type, and some in addition had twiglike or basketlike axon terminals. All cell types made synapses on distal dendrites; a proportion of the fast‐spiking and burst‐firing cells in addition had basketlike terminals that made synaptic contacts on proximal dendrites and on somata. Two morphological types of somata were postsynaptic to the basket cells: large (20–30‐μm) oval cells with dark cytoplasm, and large oval cells with paler cytoplasm, often with an apical dendrite. The presence of lamellar bodies in the large dark neurons suggests that they may be cholinergic neurons, because previous work has localised these structures in some neurons that stain for choline acetyltransferase. Our work suggests therefore that there may be GABAergic neurons in the MS/DB that form basket synaptic contacts on at least two types of target cell, possibly cholinergic and GABAergic neurons, which means that the basket cells could play a key role in the generation of rhythmic activity in the MS/DB. J. Comp. Neurol. 430:410–432, 2001.

Conduction velocities and membrane properties of different classes of rat septohippocampal neurons recorded in vitro

1 The membrane properties and conduction velocities of antidromically activated medial septum‐diagonal band (MS‐DB) neurons were examined using whole‐cell recordings in a longitudinally cut rat brain slice preparation containing the MS‐DB and the dorsal fornix. 2 MS‐DB neurons were divided into three groups according to their action potential characteristics and firing properties. Slow firing neurons displayed a broad action potential followed by a prominent after‐hyperpolarization. Burst firing neurons, when depolarized from hyperpolarized holding potentials, exhibited a high‐frequency burst of spikes on the crest of a slow depolarizing potential. Fast firing neurons did not fire bursts of spikes when depolarized from hyperpolarized holding potentials. 3 Eighteen MS‐DB neurons were identified as septohippocampal by antidromic activation. Of the septohippocampal neurons, four were slow firing neurons, five were burst firing neurons and nine were fast firing neurons. The mean axon conduction velocities of these neurons fell into two significant groups, termed slow conducting and fast conducting. Slow firing septohippocampal neurons had significantly slower conduction velocities than either fast firing or burst firing neurons (P < 0.05), being 0.7 ± 0.5 ms−1 for slow firing neurons and 2.9 ± 2.0 and 2.0 ± 1.4 ms−1 for burst firing and fast firing neurons, respectively. 4 On the basis of previous evidence which has linked firing properties with the neurochemical identities of the neurons, we propose that the slow firing septohippocampal neurons are cholinergic whereas the burst firing and fast firing septohippocampal neurons are GABAergic.

A parvalbumin-containing, axosomatic synaptic network in the rat medial septum: relevance to rhythmogenesis*

The medial septal diagonal band complex (MS/DB), made up of cholinergic and GABAergic neurons, plays an important role in the generation of the hippocampal theta rhythm. A GABAergic neuron type in the MS/DB that has fast spiking properties was shown previously to contain parvalbumin immunoreactivity and to form axosomatic connections with unidentified somata. The aim in the current study was to determine the neurochemical identities of these target neurons. In slices and in perfused‐fixed brain, staining for parvalbumin immunoreactivity first of all revealed the presence of two types of parvalbumin‐positive somata in the MS/DB: medially located neurons with parvalbumin‐positive basket‐like terminals on them, and more laterally located neurons with fewer parvalbumin‐positive contacts on them. In MS/DB slices, the terminals of fast spiking neurons filled with biocytin correspondingly made either numerous contacts that surrounded the parvalbumin‐positive cell body in basket‐like formation, or 1–5 contacts on a localized patch of the soma. These contacts were shown by electron microscopy to form synaptic junctions. No terminals of biocytin‐filled fast spiking neurons were observed on cholinergic neurons, and dual staining in perfused‐fixed brain did not reveal the presence of parvalbumin‐containing terminals on cholinergic somata. Our results suggest therefore that there are two subtypes of parvalbumin‐containing neuron in the MS/DB, and that these are interconnected via axosomatic synapses. The contrasting topographical organization of the two types of parvalbumin‐containing neuron suggests that they may receive different types of afferent input, but this will require substantiation in future studies. We propose that generation of rhythmic activity in the MS/DB is controlled by contrasting contributions from two types of parvalbumin‐positive neuron, and that the role of the cholinergic neuron is modulatory.