Kainate-Induced Degeneration of Hippocampal Neurons. Protective Effect of Activation of the Endocannabinoid System.

We studied the prolonged action of kainic acid on glutamatergic neurons in the dorsal hippocampus and the endocannabinoid-dependent protection against neurodegeneration. The pyramidal neurons of the CA3 field of the hippocampus, as well as granular and mossy cells of the dentate gyrus were examined. Light and electron microscopy revealed substantial damage to the components of the protein-synthesizing (rough endoplasmic reticulum, Golgi apparatus, and polyribosomes) and catabolic (lysosomes, autophagosomes, multivesicular structures, and lipofuscin formations) systems in all cells. Pyramidal and mossy neurons die mainly by the necrotic pathway. The death of granular cells occurred through both apoptosis and necrosis. The most vulnerable cells are mossy neurons located in the hilus. Activation of the endocannabinoid system induced by intracerebral injection of URB597, an inhibitor of degradation of endocannabinoid anandamide, protected the normal structure of the hippocampus and prevented neuronal damage and death induced by KA.

Ultrastructural Alterations in Granular Neurons of the Dentate Fascia Caused by Intrahippocampal Injection of Beta-Amyloid 1-42.

The deposition of beta-amyloid (Aß) in the brain is detected in Alzheimer's disease and during ageing. Until now, ultrastructural studies of changes caused by Aß in the dentate gyrus are very scarce. The effects of Aß 1-42 injection into the CA1 field of rat hippocampus were studied by electron microscopy. In 2 weeks after injection of aggregated Aß in low concentrations, destructive changes were seen in the structure of dentate gyrus cells, which consisted in a decrease in the number of dentate gyrus neurons and axo-dendritic synapses. These changes were accompanied by enlargement of the endoplasmic reticulum cisterns and widening of the active zones of synapses. Thus, injection of aggregated Aß 1-42 into the hippocampus led to irreversible (a decrease in the number of neurons and axo-dendritic synapses, agglutination of synthetic vesicles) and adaptive changes (an increase in the sizes of endoplasmic reticulum cisterns and active zones of synapses) in dentate gyrus neurons aimed at the maintenance of functional activity of the nervous system.

[Endogenous Cannabinoid System of the Brain as the Target for Influences at Neurodegenerate Diseases].

The review represents the analysis of works about role of endogenous cannabinoid (EC) system in the neuro- degenerate diseases (ND), in which the cellular death and disturbances of neuronal functions of the hippo- campus, neocortex and striatum are observed. Here, the diseases.ofAlzheimer, of Parkinson, of Hangtington, and the temporal lobe epilepsy are considered. Data, on the known or assumed mechanisms of these diseases are provided. In spite of the fact that the etiology.of the listed ND varies, their pathogenesis is characterized by common features: neural hyperexcitability, trophic deprivation, oxidative stress and energy deficite. Cognitive violations and/or deficiency of senso-motor integration are characteristic of patients with ND. Now reliable medicines for treatment of ND are absent. In recent years the fundamental role of EC system in regu- lation of neuroexcitability, energy metabolism, inflammatory and many other processes has been opened in ND pathogenesis. It points to possibility of development of therapeutic approaches which use the prepara- tions for activation of EC system. In the review various mechanisms of cellular survival and their reparations provided to EC system during action of pathological factors are stated.

Network effects of glutamate on neuronal activity in the medial septum/diagonal band complex in vitro.

Inter-neuronal interactions within the medial septum/diagonal band complex (MSDB) are of great interest as this region is believed to be the hippocampal theta rhythm pacemaker. However, the role of glutamatergic system in functioning of the septal cells is yet unclear. Here, we demonstrate for the first time the effects of glutamate in physiological concentration (1 microM) on the MSDB neuronal spontaneous and evoked activities in vitro. These effects (activation of 70% and inhibition of 30% of responsive neurons) differed in pacemaker and non-pacemaker cells. Pacemaker cells were always activated under glutamate, whereas non-pacemaker neurons could be either activated or inhibited. Indeed, in the burst pacemakers, glutamate increased the frequency of rhythmic activity. In a total MSDB neuron population, in 30% of neurons glutamate applications modified responses to the electrical stimulation by unifying the temporal parameters of neuron responses. Along with the increase in the theta-burst frequency, this indicates that the glutamatergic system is involved in the process ofintraseptal synchronization. Obtained data shed light on the role ofglutamatergic system in septal neuron interactions and broaden our understanding of theta oscillation mechanisms in the septo-hippocampal system.

Modulation of theta rhythmicity in the medial septal neurons and the hippocampal electroencephalogram in the awake rabbit via actions at noradrenergic alpha2-receptors.

The modulation of the firing discharge of medial septal neurons and of the hippocampal electroencephalogram (EEG) mediated by actions on alpha2-adrenoreceptors (ARs) was investigated in awake rabbits. Bilateral i.c.v. infusion of a relatively low dose (0.5 microg) of the alpha2-AR agonist clonidine produced a reduction in the theta rhythmicity of both medial septal neurons and the hippocampal EEG. In contrast, a high dose of clonidine (5 microg) increased the percentage and degree of rhythmicity of theta bursting medial septal neurons as well as the theta power of the hippocampal EEG. On the other hand, administration of alpha2-AR antagonist idazoxan produced the opposite dose-dependent effect. While a low dose of the antagonist (20 microg) produced an increase in both the theta rhythmicity of medial septal neurons and the theta power of the hippocampal EEG, a high dose (100 microg) caused a reduction of theta rhythmicity in both the medial septum and hippocampus. These results suggest that low doses of alpha2-ARs agents may act at autoreceptors regulating the synaptic release of noradrenaline, while high doses of alpha2-ARs drugs may have a predominant postsynaptic action. Similar results were observed after local injection of the alpha2-AR drugs into the medial septum suggesting that the effects induced by the i.c.v. infusion were primarily mediated at the medial septal level. We suggest that noradrenergic transmission via the postsynaptic alpha2-ARs produces fast and strong activation of the septohippocampal system in situations that require urgent selective attention to functionally significant information (alert, aware), whereas the action via the presynaptic alpha2-ARs allows a quick return of the activity to the initial level.

Neuronal activity of the septal pacemaker of theta rhythm under the influence of stimulation and blockade of the median raphe nucleus in the awake rabbit.

The control of theta rhythm in neuronal activity of the medial septal area and hippocampal electroencephalogram by the brainstem structures was investigated in waking rabbits. In the first series of experiments stimulating electrodes were implanted into the midbrain reticular formation and median raphe nucleus. The standard frequency of theta-bursts in medial septal area neurons and in the electroencephalogram was uniformly and chronically decreased in all rabbits with electrodes implanted into the median raphe nucleus (4.7 +/- 0.5 Hz versus 5.2 +/- 0.19 Hz in animals without electrodes in median raphe nucleus). Weak electrical stimulation of the median raphe nucleus resulted in additional decrease of theta expression in the medial septal area neurons and its disappearance from the hippocampal electroencephalogram, where it was substituted by delta-waves and spindles. Stimulation of the reticular formation had the opposite effect, with an increase in theta frequency, regularity and expression in medial septal area neuronal activity and hippocampal electroencephalogram. In the second series of experiments reversible functional blockade of the median raphe nucleus by local microinjection of lidocaine was performed. This resulted in expression of theta-bursts in an additional group of medial septal area neurons, an increase in theta-burst frequency (by 0.5-2 Hz) and regularity with concomitant changes in the electroencephalogram. The effects of sensory stimuli on the background of increased theta activity were suppressed or significantly decreased. It is concluded that, in accordance with the data of other authors, the median raphe nucleus can be regarded as a functional antagonist of the reticular formation, powerfully suppressing theta-bursts of the medial septal area neurons and hippocampal theta rhythm. It is suggested that, in combination with the theta-enhancing influences of reticular formation, the median raphe nucleus may participate in termination of attention, its switching to other stimuli and stabilization of the effects of learning.

Spontaneous activity and sensory responses of hippocampal neurons during persistent theta-rhythm evoked by median raphe nucleus blockade in rabbit.

Spontaneous activity and responses to sensory stimuli were analysed in the hippocampal CA1 neurons of chronic unanesthetized rabbits before and after reversible functional blockade of the median raphe nucleus and medial septal area by local microinjections of anesthetic lidocaine. This evoked, correspondingly, persistent theta rhythm and its complete blockade for about 30 min. The results were compared to the neuronal data obtained earlier in the experiments with cholinergic drugs modulating expression of theta rhythm. Intra-median raphe nucleus injection of lidocaine evoked uniform increase of discharge rate in the hippocampal neurons with low and high spontaneous activity. Theta modulation of neuronal activity had increased regularity and frequency (by 0.5-2.0 Hz) and appeared in additional group of the neurons simultaneously with expression of persistent theta in the hippocampal electroencephalogram. Sensory responsiveness of the hippocampal neurons was drastically decreased (45% of the responses preserved). Reactions of all types were blocked, diminished, or inverted, but inhibitory responses were the most severely affected. Injection of lidocaine into medial septal area also blocked all brain stem afferents ascending to the hippocampus via medial septal area, thus, totally depriving hippocampus of brainstem-septal input. However, besides the total absence of theta modulation, spontaneous activity in majority of neurons was not significantly changed. Responsiveness to sensory stimuli also remained relatively high (77% of the responses preserved); on-effects were especially resistant to medial septal area blockade. Comparison of spontaneous and evoked activity in two theta states (physostigmine and median raphe nucleus blockade) revealed striking similarity of all characteristics, which suggested that theta-suppressing influences of median raphe nucleus (presumably serotonergic) are realized primarily through the control of cholinergic septo-hippocampal theta-generating mechanism. However, as the frequency of theta rhythm does not depend on it, an additional effect of disinhibition of activating reticular formation by the median raphe nucleus suppression is suggested. The data confirm that theta rhythm may be regarded as active filter in the information processing by the hippocampal neurons.

Pacemaker neurons of the forebrain medical septal area and theta rhythm of the hippocampus.

The hippocampus is of critical importance for the organization of selective attention and memory. The activity of its neurons is rhythmically modulated by the direct afferent input from cholinergic and GABAergic neurons of the medial septal nucleus and the nucleus of diagonal band (MS-DB). This modulation is expressed in the hippocampal electroencephalogram as a slow (4-9 Hz) sinusoidal theta rhythm. The paper presents a short review of the authors' data on the properties of MS-DB cells, their interactions in organizing the theta expression in the hippocampus, and the probable functional significance of this rhythm. Extracellular recordings of neural activity of the MS-DB and hippocampus were performed in chronic alert rabbits with transection of various afferent pathways, in brain slices, and in the embryonic tissue grafts developing in the anterior eye chamber and brain. Theta modulation disappeared in all structures deprived of the MS-DB afferent connections, but was retained by MS-DB neurons under isolation conditions. A limited group of MS-DB neurons (6-8%) discharge in regular bursts after complete synaptic blockade in high Mg(2+)-low Ca2+ medium, while other neurons in these conditions exhibited the highly regular single-spike activity. Thus, the MS-DB neurons can be regarded as bursting and regular pacemakers. The frequency of bursts and the number of neurons involved in the rhythmic activity are in parallel increased by afferent stimulation-either natural (sensory stimuli) or imitated by electrical stimulation of ascending reticular formation. An increase in the number of MS-DB neurons secondarily involved in theta bursts with the resulting expression of theta rhythm in EEG can be also obtained by up-regulation of endogenous acetylcholine (by physostigmine), but in this case the frequency of theta is not changed. The MS-DB neurons contain acetylcholine and GABA as neurotransmitters. Analysis of the effects of their agonists and antagonists suggests that the frequency of theta depends on the GABAergic mechanism, while its power is controlled by cholinergic influences. In the control state, input signals triggered in the hippocampal neurons an inhibitory reset followed by synchronous theta modulation, gradually habituating during repeated presentations of the stimulus. Against the background of continuous theta evoked by physostigmine, the responses were blocked or significantly depressed, while after theta suppression by scopolamine the efficacy of the stimuli was increased, and habituation was absent. It is suggested that the theta rhythm operates as a selective filter: it augments and prolongs the input signal by which it was triggered and simultaneously protects it from the interference of extraneous stimuli appearing during its processing and registration. The theta rhythm may be regarded as an important mechanism of selective attention, which is a prerequisite for memory trace formation.

Acetylcholine, theta-rhythm and activity of hippocampal neurons in the rabbit--I. Spontaneous activity.

The background activity of hippocampal neurons was recorded extracellularly in waking rabbits in the control state and after systemic injections of physostigmine and scopolamine. Similar analysis was done in the hippocampus chronically deprived of ascending brainstem afferents. Cholinergic drugs control the number of hippocampal neurons with theta modulation and stability, but not the frequency of theta modulation. Increase of endogenous acetylcholine also resulted in regularization of the activity with suppression of delta modulation and complex spike discharges; its blockade produced the opposite changes. Both drugs changed the level of background activity in the majority of the neurons, but the overall mean frequency did not vary between the states. Regression analysis demonstrated significant negative correlations with dominating decrease in the level of discharges in high-frequency neurons (> 25 spikes/s) and its increase in low-frequency ones (< 25 spikes/s) after injection of both drugs. Stability of the overall mean frequency and uniformity of its shifts during both stimulation and suppression of the cholinergic component of theta-rhythm presumably indicate that the frequency of background activity, unlike its pattern, is not directly controlled by the cholinergic septal input.

Acetylcholine, theta-rhythm and activity of hippocampal neurons in the rabbit--II. Septal input.

The aim of this paper was to evaluate the cholinergic component of the septohippocampal input signals in neuronal activity of the hippocampal fields CA1 and CA3 recorded extracellularly in chronic alert rabbits. Effects of electrical stimulation of the medial septal area were analysed in the control state, on the background of an increased level of endogenous acetylcholine (by physostigmine injection) and during its blockade by antimuscarinic drugs (scopolamine, atropine). Two groups of animals were used in the experiments: intact rabbits and rabbits with complete chronic undercutting of the septum, depriving the septohippocampal system of ascending medial forebrain bundle afferents. Primary inhibitory effects of standard duration (40-140 ms) evoked by medial septal area stimulation dominated in the hippocampus of intact rabbits (54%), though some neurons responded by initial diffuse excitation (37.5%); responses by single-spike on-effects were observed in a minority of neurons (8.5%). The primary suppression of activity prevailed (90%) in animals with basal undercutting of the septum. In intact rabbits under physostigmine action, the effects of medial septal area stimulation were depressed or completely blocked in 78% of hippocampal neurons on the background of increased theta modulation of activity. Neuronal responses to medial septal area stimulation recovered at the background of muscarinic antagonists. These effects of cholinergic drugs were reproduced in animals without medial forebrain bundle. It is concluded that the initial effect of the septal input upon the hippocampal neurons consists of a general suppression of their activity (reset), depending upon a non-cholinergic (presumably GABAergic) component of the septohippocampal connections.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine, theta-rhythm and activity of hippocampal neurons in the rabbit--III. Cortical input.

Cholinergic modulation of single cell responses and field potentials evoked in the hippocampus by electrical stimulation of the perforant path and mossy fibres was investigated in two groups of chronic unanesthetized rabbits--with intact hippocampus and with basally undercut septum (without ascending medial forebrain bundle afferents). In both groups of animals responses to stimulation were blocked or significantly depressed by i.v. physostigmine injection in many neurons (50% in the intact hippocampus and 69% in the hippocampus without medial forebrain bundle). In minor groups of neurons (10 and 8%, respectively), facilitation of responses was observed. Scopolamine restored initial responsiveness of hippocampal neurons and augmented effects of stimulation in some of them. The effect of physostigmine was reproduced by stimulation of the medial septum. Depressive influence of medial septal area stimulation was increased by physostigmine and blocked by scopolamine. Population spikes evoked by stimulation of the perforant path of the intact group were equally suppressed (by 43%) during sensory stimulation evoking natural theta, after physostigmine and after medial septal area stimulation. In the group of animals without medial forebrain bundle these influences resulted in a complete suppression of field potentials; scopolamine restored them. It is concluded that the main function of the septohippocampal cholinergic input consists of filtering out the signals appearing at the background of theta-rhythm triggered by a previous signal, thus preventing their interference with its processing and registration.

Acetylcholine, theta-rhythm and activity of hippocampal neurons in the rabbit--IV. Sensory stimulation.

Modifications of responses of hippocampal neurons to sensory stimuli at the background of increased endogenous acetylcholine level (injection of physostigmine) and during blocking by scopolamine were analysed in the chronic alert rabbit. A significant decrease of reactivity (about 40%) of hippocampal neurons to sensory stimuli occurred after physostigmine injection, inducing stable theta modulation. Suppression and decrease of inhibitory responses (including initial reset phase) and of some excitatory reactions (including on-effects) were observed. However, a limited group of excitatory responses was augmented and prolonged under physostigmine action. Scopolamine, which blocked electroencephalogram theta-rhythm, did not change the responsiveness of hippocampal neurons. Some of the inhibitory and excitatory effects of sensory stimuli, especially on-responses, were strongly facilitated. Tonic responses were shorter, but they were stably reproduced without typical gradual habituation. All these effects were also present in the hippocampus after basal undercutting of the septum, which eliminates ascending brainstem input. It is suggested that under normal conditions a new or significant sensory stimulus evokes, in the hippocampus, an initial inhibitory reset of neuronal activity with subsequent coordinated triggering of rhythmic theta modulation by the septal input and arrival of the cortical input signal phase-locked to it. During the period of theta triggered by the stimulus, its processing and fixation in memory occurs, while the other, interfering stimuli, which are not phase-locked to the ongoing theta activity, are actively filtered out. Thus, septohippocampal interactions may participate in the organization of selective attention as a necessary condition for memory trace formation.

Neuronal activity of the septum transplanted into the neocortical barrel field of the rat.

Embryonic (E16-17) septal solid grafts were transplanted into acute cavities in the barrel field of somatosensory neocortex of adult rats. Extracellular recording of the graft's neuronal activity was performed in lightly anesthetized rats, or in brain slices in vitro 8-10 months after grafting. Analysis of Nissl and Golgi-Cox stained preparations showed that 81% of the grafts survived. Judging by combined histological and electrophysiological criteria, 69% of surviving grafts were integrated with the host brain. All septal grafts contained neurons with high spontaneous activity (mean 14.9 ± 8.3 spikes · s -1). Irregular bursts or rhythmic theta-bursts were present in background activity. The frequency of theta-bursts varied in parallel with the state of the animal from 3 Hz (deep barbiturate sleep) up to 7-8 Hz (arousal). Somatosensory stimulation evoked initial bursts or suppression of activity, often followed by a period of rhythmic theta-bursts. Though a high level of convergence for stimulation of vibrissae and body surface was typical of the grafted neurons, a certain spatial gradient of body representation was present. Many neurons responded also by initial burst or prolonged suppression of activity to electrical stimulation of thalamus and homolateral motor cortex. In the frontal slices of neocortex the neurons of septal grafts were highly reactive to stimulation of adjacent neocortex, but usually when the distance between the stimulating electrodes and the graft border did not exceed 1-1.5 mm. The data obtained in the septal slices are compared to the results described earlier in homotopic neocortical and heterotopic hippocampal grafts to the barrel field. It is concluded that heterotopic embryonic septal grafts can successfully develop and establish functional afferent connections with the host brain. Their neurons can participate in processing of sensory information, appropriate to the substituted cortical area, though the characteristics of responses are to a great extent determined by intrinsic properties of the septal neurons.

Functional integration of the rat hippocampal tissue, transplanted into the rabbit septum.

Embryonal tissue of the rat hippocampus (E17-18) was grafted into small acute cavity within the dorsal septum of adult rabbit after interruption of septo-hippocampal connections. Histological analysis (Nissl stain), performed 6-8 weeks later, revealed surviving hippocampal grafts in 14 out of 29 grafted animals (48%). Some of the grafts were displaced into the ventricle and had only minor contact with the host septum; some others, residing in the septum, were separated from the host tissue by continuous glial scar. Six grafts (33%) were morphologically well integrated with the host septum. The grafts contained mature pyramidal neurons but their organization into layer was absent. No signs of rejection were observed in the grafts, which were not subjected to microelectrode investigation in chronic conditions. The grafts, which for 5-9 successive days were penetrated by microelectrodes, were heavily infiltrated by lymphocytes and had necrotic areas. Electrophysiological testing revealed the absence of spontaneous neuronal activity, or the presence of hypersynchronous epileptiform bursts in isolated grafts. The 4 integrated grafts, investigated electrophysiologically, contained neurons with normal spontaneous activity of two types: low-frequency discharges with complex spikes and high-frequency single spikes, sometimes with short periods of weak rhythmic modulation in the theta-range. Characteristics of spontaneous activity of the xenografted hippocampus are very close to those described in the rat hippocampus in situ. Rhythmic theta-modulation of neuronal activity appeared or increased in stability after physostigmine injection to the host, after electrical stimulation of the host's midbrain reticular formation and ventral part of diagonal band, and after multimodal sensory stimulation of the host. The frequency of theta-bursts in the grafts was identical to that of neurons of the host septum (4.5-6.5 Hz). Besides the theta-modulation sensory stimuli evoked tonic suppression or activation of discharges, phasic reactions and on-effects. Electrical stimulation of lateral septum and diagonal band evoked orthodromic and antidromic discharges of the grafted cells. Normal spontaneous and evoked activity of the grafted cells deteriorated after 5-11 successive days of microelectrode investigation. It is concluded that xenografts of nervous tissue, developing without immunosuppression, have a low rate of survival and are vulnerable to immune response after mechanical interruption of integrity of the blood-brain barrier. Nevertheless their normal activity and close functional integration with the host brain, apparently depending on development of chimeric synapses, is possible.