Interaction of HIV Tat and matrix metalloproteinase in HIV neuropathogenesis: a new host defense mechanism.

Tat, the HIV transactivating protein, and matrix metalloproteinases (MMPs), a family of extracellular matrix (ECM) endopeptidases, have been implicated in the pathogenesis of HIV-associated dementia. However, the possibility that MMPs interact with viral proteins has remained unexplored. We therefore treated mixed human fetal neuronal cultures with recombinant Tat and select MMPs. Neurotoxicity was determined by measuring mitochondrial membrane potential and neuronal cell death. Previous studies have shown that Tat and MMP independently cause neurotoxicity. Surprisingly, we found the combination of Tat and MMP produced significant attenuation of neurotoxicity. To determine whether there was a physical interaction between Tat and MMP, we used protein electrophoresis and Western blot techniques, and found that MMP-1 can degrade Tat. This effect was blocked by MMP inhibitors. Furthermore, MMP-1 decreased Tat-mediated transactivation of the HIV long terminal repeat region, and this functionality was restored when MMP-1 activity was inhibited. These results suggest that the decrease in Tat-induced neurotoxicity and HIV transactivation is due to Tat's enzymatic cleavage by MMP-1. The direct interaction of human MMPs with viral proteins has now been demonstrated, with resultant modulation of Tat-mediated neurotoxicity and transactivation. This study elucidates a unique viral-host interaction that may serve as an innate host defense mechanism.

Molecular and cellular mechanisms of neuronal cell death in HIV dementia.

The deaths of neurons, astrocytes and endothelial cells have been described in patients with HIV (human immunodeficiency virus) dementia. HIV-1 does not infect neurons; instead, neurotoxic substances shed by infected glia and macrophages can induce a form of programmed cell death called apoptosis in neurons. These neurotoxins include the HIV-1 proteins Tat and gp120, as well as pro-inflammatory cytokines, chemokines, excitotoxins and proteases. In this article we review the evidence for apoptosis of various cell types within the brain of HIV-infected patients, and describe in vitro and in vivo experimental studies that have elucidated the mechanisms by which HIV causes apoptosis of brain cells.

Differential transcriptional regulation by human immunodeficiency virus type 1 and gp120 in human astrocytes.

Astrocytes may be infected with the human immunodeficiency virus type 1 (HIV-1) or exposed to the HIV protein gp120, yet their role in the pathogenesis of HIV dementia is largely unknown. To characterize the effects of HIV on astrocytic transcription, microarray analysis and ribonuclease protection assays (RPA) were performed. Infection of astrocytes by HIV or treatment with gp120 had differential and profound effects on gene transcription. Of the 1153 oligonucleotides on the immune-based array, the expression of 108 genes (53 up; 55 down) and 82 genes (32 up; 50 down) were significantly modulated by gp120 and HIV infection respectively. Of the 1153 oligonucleotides on the neuro-based array, 58 genes (25 up; 33 down) and 47 genes (17 up; 30 down) were significantly modulated by gp120 and HIV infection respectively. Chemokine and cytokine induction occurred predominantly by HIV infection, whereas gp120 had no significant effect. These results were confirmed by RPA. The authors conclude that profound alterations of astrocytic function occur in response to HIV infection or interaction with viral proteins, suggesting that astrocytes may play an important role in the pathogenesis of HIV dementia.

Intraseptal injection of scopolamine increases the effect of systemic diazepam on passive avoidance learning and emotionality in rats.

This experiment was designed to assess the role of the septo-hippocampal cholinergic (ACh) system in the deleterious effects produced by systemic benzodiazepine injection on learning processes in rats. Retention of a step through passive avoidance task was analysed after systemic injection of increasing doses of either scopolamine or diazepam applied alone 30 min before the acquisition phase. Results indicated a dose related impairment of retention by each drug: in addition, sub-threshold doses of scopolamine and diazepam applied in combination (diazepam: 2mg/kg plus scopolamine: 0.3mg/kg) produced a decrease of retention latencies, thus showing an additive effect of the combined treatment. Secondly, a sub-threshold dose of scopolamine (15microg/0.5microl) was also administered into the medial septal area, together with an i.p. injection of 2mg/kg of diazepam. This combined treatment produced a severe impairment of retention, in parallel with a large reduction in emotionality (number of faeces). The data are consistent with the hypothesis that peripheral administration of behaviorally effective doses of diazepam on passive avoidance learning might act partially via a septal ACh-GABA/benzodiazepine mechanism. It is also suggested that this mechanism subserves both anxiety and the memorisation of contextual stimuli associated with passive avoidance acquisition, through the modification of the septo-hippocampal activity.

Differential involvement of the lateral and medial divisions of the septal area on spatial learning processes as revealed by intracranial self-administration of morphine in mice.

The present study was conducted to investigate the effects of morphine self-administration into either the medial or lateral divisions of the septal region on spatial discrimination abilities in mice. To this end, BALB/c mice received a dose of 5 ng/50 nl of morphine sulfate, via a stainless steel injection cannula, inserted into either the medial septal area (MS) or the lateral septal nucleus (LS), when they are close to the end of one of the two choice arms of a Y-maze (acquisition period). In these conditions a discrimination between the reinforced arm and the neutral arm is acquired in MS as well as in LS mice. However both acquisition and subsequent extinction (vehicle only available) was more rapid in the LS group than in the MS group. Moreover, during the extinction period, numerous escape attempts from the Y-maze were observed for MS mice. When the dose of morphine was raised to 50 ng the pattern of acquisition was unchanged in the LS group. In contrast animals of the MS group learned to avoid the arm where the higher dose of morphine was delivered thus allowing us to conclude that in MS animals the drug effect became aversive at this higher dose. This possibility was directly investigated in a second experiment by closing the access to the neutral arm. Thus, for a 5-ng dose the rewarding effect of morphine was demonstrated in both MS and LS groups by the decrease of self-administration latencies which, furthermore, were notably lower than in the discrimination situation. Moreover, with the dose of 50 ng of morphine the latencies were identical for the two groups and at their lowest value thus indicating that morphine had similar appetitive effects in MS as well as in LS mice in this situation. Thus, the previously observed deficit of MS subjects, including escape attempts, disappeared when the dose of morphine was raised and the experimental context simplified. These results which demonstrate differential functional properties of these two septal divisions are discussed in terms of conflict resulting from the strongly appetitive effects of the morphine which induces, in parallel, deleterious consequences on cognitive processing in MS self-injected mice.

Modulation of spatial alternation and anxiety by septal scopolamine systemic diazepam in mice.

To investigate the behavioral consequences of benzodiazepines in subjects whose septo-hippocampal cholinergic (ACh) activity was impaired, C57BL/6 mice received an injection of 2.5 microg/0.2 microl of scopolamine into the medial septal area with an i.p. injection of 0.5 mg/kg of diazepam. The consequences of these treatments administered in combination or alone were evaluated on anxiety measured in an elevated plus-maze and on spontaneous alternation carried out in a T-maze, using two different intertrial intervals (ITI: 5s or 30s). In these conditions, only the combined treatment provoked a decrease of the anxiety level, which was associated with an impairment of spontaneous alternation restricted to the 5s ITI. Because mice were not impaired during the sequential 30s ITI, this seems to rule out the possibility that this alternation deficit resulted from a working memory loss. These results suggest an involvement of a septal ACh-GABA-A/BDZ interaction in the exaggeration of cognitive deficits produced by benzodiazepines in patients characterized by a cholinergic hypofunction.

Septal scopolamine-systemic diazepam interaction in passive avoidance learning in rats.

The role of the septal region in passive avoidance (step-through) learning was studied by administering 15µg/0.5µl of scopolamine intraseptally to rats, together with an i.p. injection of 2mg/kg of diazepam to the same animals. The combined treatment provoked a severe impairment in retention of the learning situation, whereas animals which received either scopolamine or diazepam alone did not show any deficit.

Consequences of selective blockade of septal noradrenergic afferents on anxiety and spatial working memory performance in mice.

This experiment was designed to investigate the role of septal noradrenergic (NA) afferents in the control of anxiety and spatial working memory. To this end, C57Bl/6 mice were infused bilaterally into the lateral septal nuclei with 500 ng/0.2 microliter of BE 2254, a selective alpha 1 postsynaptic adrenoceptor antagonist. The consequences of this reversible treatment were evaluated 20 min later on the anxiety level measured in an elevated plus-maze and on spatial working memory, evaluated under four different conditions via the learning of a delayed nonmatching to place (DNMTP) rule achieved in an eight-arm radial maze. In these conditions, the BE 2254, as well as the saline-injected control group, showed an elevation of the anxiety level that may be the indirect expression of a nonspecific septal dysfunction induced by the vehicle injection rather than the normal behavioral response produced by the decrease of septal NA activity. This septal dysfunction also impaired spatial working memory but only when mnesic difficulty of the task is increased, suggesting that this impairment expresses a general memory deficit rather than a working memory deficit per se. A lack of spatial working memory deficits in BE 2254 or saline-injected animals was also observed in two other conditions of the behavioral protocol. However, when treatments were applied before the first exposure of animals to the radial maze (exploration session), only the group which received BE 2254 was impaired during the acquisition session for the rule performed 24 h later. This delayed perturbation seems to be linked, at this stage of the learning procedure, to the lack of NA-dependent processes taking place during the exploration session. Taken together, these data suggest that septal NA mechanisms are more essential at initial stage of this learning, when animals process new features of the situation, than during the expression of spatial working memory per se.

Relationships between septo-hippocampal cholinergic activation and the improvement of long-term retention produced by medial septal electrical stimulation in two inbred strains of mice.

This experiment was designed to highlight the relationships between septo-hippocampal cholinergic activation and the processing of memory consolidation. For that purpose, we have analyzed the consequences of a medial septal electrical stimulation (100 Hz, 30 microA) applied soon after partial acquisition session of an appetitive operant conditioning task on in vivo hippocampal cholinergic activity on the one hand and on subsequent retention 24 h later on the other hand. For maximize our data base for such comparison we used two neurochemically and behaviorally distincts strains of mice, BALB/c and C57BL/6. In these conditions, our results showed that BALB/c mice evidenced better performance in retention than C57BL/6 mice after medial septal stimulation. On the other hand, the stimulation applied in resting conditions produced a moderate and similar hippocampal cholinergic activation in the two strains. Moreover, in BALB/c mice the only strain which exhibited good consolidation capacities the stimulation do not induced any additive effect on the substantial increase of the hippocampal cholinergic activity produced by the previous acquisition session. Finally, a correlative study realized in C57BL/6 mice seems to indicate that the higher the hippocampal cholinergic activity was the lower were the consolidation capacities. These results lead us to suggest that the improvement of memory consolidation induced by the medial septal stimulation is the consequence of the recruitment of non cholinergic elements located close to the electrode tip. Consequently the septo-hippocampal cholinergic activation is more likely to facilitate certain information processes prior to the consolidation mechanisms proper.

Post-training medial septal stimulation improves spatial information processing in BALB/c mice.

The experiment reported here was designed to highlight the effects of medial septal nucleus electrical stimulation on memory processes. To that end, we have used a learning task carried out in a 4-hole board in which animals were successively presented with the location of 2 baited holes. Under these conditions, the consequence of a weak stimulation (30 microA peak to peak, 80 s in duration) applied 30 s after acquisition of these 2 separate spatial informations was assessed on a retention test 24 h later. The results indicated that performance of stimulated animals was improved by medial septal stimulation as shown by an increase in the time duration of exploration of the second previously baited hole. However, post-training stimulation also produced a significant avoidance of the first previously baited hole not observed in the control groups. These results suggest that medial septal stimulation improves retroactive inhibition involved in working memory processes, thus leading to better retention of this kind of learning situation.

The intermediate stage of sleep in mice.

Seven mice of Balb/C strain were implanted with electrodes to perform sleep-waking recordings. In 100% of the cases, the mice showed, prior to paradoxical sleep, the intermediate stage of sleep characterized by high-amplitude cortical spindles interspersed with slow waves and low-frequency theta rhythm in the dorsal hippocampus. Consequently, the intermediate stage which seems to correspond to a transient functional isolated forebrain does exist in the rat, cat and mouse. in the rat, cat and mouse.

In vivo modulation of septo-hippocampal cholinergic activity in mice: relationships with spatial reference and working memory performance.

Dopaminergic afferents to the septum mediate a tonic and trans-synaptic inhibitory control on the cholinergic neurones of the septo-hippocampal pathway. Lesion of these afferents using 6-hydroxydopamine (6-OHDA) results in a chronic and specific increase of hippocampal cholinergic activity in mice. The consequence of this in vivo modulation of hippocampal cholinergic activity on the acquisition of both a spatial discrimination and a working memory (delayed non-matching to place) task in an 8-arm radial maze by C57BL/6 mice were investigated. Combined neurochemical and behavioural analyses revealed significant correlations between hippocampal sodium-dependent high-affinity choline uptake activation induced by testing and performance measures. In the first experiment 6-OHDA-treated mice compared to control and vehicle-injected mice showed a transient (day 2) but significant facilitation of their spatial discrimination performance which appears to be better related to the working but not to the reference memory component of the task. This hypothesis is strengthened by the results of the second experiment which shows an amelioration of working memory performance when the septo-hippocampal cholinergic pathway is specifically activated in vivo.

Differential hippocampal and cortical cholinergic activation during the acquisition, retention, reversal and extinction of a spatial discrimination in an 8-arm radial maze by mice.

Possible differentiation of the intervention of cholinergic septohippocampal and magnocellular forebrain (NBM) projections to cortex during learning and memory processes has been investigated directly using mice. High-affinity choline uptake velocities in the hippocampus and cortex were analyzed, in parallel, at various periods during the acquisition, over 8 days, as were the subsequent retention, reversal and extinction of a spatial discrimination in an 8-arm radial maze. Initial acquisition induced an immediate (30 s) and long-lasting (approx. 3 h) increase in mean hippocampal (+33%) and cortical (+23%) cholinergic activities. The time course of this activation was structure-dependent and correlations of hippocampal-cortical cholinergic activities showed large and consistent alterations as a function of time after training. Cholinergic activation in both brain regions was observed immediately following each daily training session with amplitudes which did not vary significantly in spite of a progressive daily increment in performance. Following acquisition mice were tested for retention, reversal and extinction: 30 s following the retention session, cholinergic activation was observed in both cortex and hippocampus, with magnitudes similar to those observed at the end of acquisition. However, in the reversal and extinction groups, a treatment-dependent attenuation of cholinergic activation was observed which was accompanied by a significant loss of correlation of cholinergic activity between these two brain regions. The results are discussed in relation to the concepts of reference and working memory and also to novelty, stress, arousal and frustrative non-reward. The data constitute direct experimental evidence for a differential involvement of cholinergic septohippocampal and NBM-cortical projections in learning and memory processes.

Electrical self-stimulation in the medial and lateral septum as compared to the lateral hypothalamus: differential intervention of reward and learning processes?

The characteristics of the electrical self-stimulation behavior elicited from the lateral hypothalamus (LH) and from both medial (MS) and lateral (LS) parts of the septal nucleus have been compared in male mice of the BALB/c strain. Using two different experimental situations (the lever-press box and a spatial discrimination test in a Y-maze) the self-stimulation rate-current intensity relations and the performances during both acquisition and reversal of a spatial discrimination were tested successively. In the lever-press box, it was observed that highest self-stimulation rates were obtained from LH placements while both MS and LS rates were lower. However, MS animals showed higher self-stimulation rates and lower self-stimulation thresholds than LS animals. Acquisition of the spatial discrimination in the Y-maze was achieved by all 3 groups with similar time courses. However, when tested for the reversal of the discrimination, the LS implanted mice were much more perturbed than the two other groups and exhibited marked perseveration. The incidence of convulsive episodes was more frequent in LS mice than in either MS or LH implanted animals during both phases of the discrimination task. These differences in the self-stimulation behavior elicited from medial and lateral parts of the septal complex are discussed in relation to the operation of positive reinforcement mechanisms and to internal inhibition processes operating during acquisition and reversal of the spatial discrimination.

The effect of diazepam on hippocampal EEG in relation to behavior.

Male mice of the BALB/cByJ and C57BL/6 strains were implanted with electrodes in the CA1 area of the hippocampus to record rhythmic slow-wave activity (RSA) or 'theta' EEG activity. The EEG spectral characteristics and the animal's motor behavior were studied while the animals walked on a moving belt (2.2 cm/s) both before and after i.p. injections of diazepam (Valium, 2 mg/kg) or vehicle. EEG spectral analyses were carried out on-line by computer. Diazepam produced a dissociation of locomotion and RSA. (1) Uninjected and vehicle-injected mice showed typical RSA (7-8 Hz) while walking. (2) Under diazepam, 7-8 Hz RSA virtually disappeared and was replaced in the temporally averaged records by RSA with a sharp, narrow-band peak at 4-5 Hz. (3) This lower-frequency RSA was associated with immobility if, and only if, the immobility immediately followed walking. This was true whether the animal itself stopped walking or the experimenter stopped the moving belt. This theta activity predominated for about 30 s and had disappeared after 2 min. Locomotion, on the other hand, was accompanied by irregular EEG activity. (4) Scopolamine (i.p. 1 mg/kg), a cholinergic blocker, greatly reduced the diazepam-induced 4-5 Hz RSA, but also partially restored 7-8 Hz RSA. The possibility that the effects of diazepam on hippocampal EEG involve changes in septohippocampal cholinergic activity is discussed.

The effects of acute intraseptal injection of haloperidol in vivo on hippocampal cholinergic function in the mouse.

Acute injection of haloperidol into the lateral septum in mice produced an immediate and long-lasting increase in hippocampal sodium-dependent high-affinity choline uptake. Parallel electrophysiological investigations revealed that the increased septo-hippocampal cholinergic activity augmented CA1 pyramidal cell excitability and also accelerated the extinction of a conditioned reinforcement. These results constitute further evidence that septal dopaminergic terminals, via their control of septo-hippocampal cholinergic activity play a significant role in the modulation of hippocampal function.

Facilitation of spontaneous and learned spatial behaviours following 6-hydroxydopamine lesions of the lateral septum: a cholinergic hypothesis.

Mice received injections of 6-hydroxydopamine (6-OHDA) in the lateral septum; they were tested for spontaneous alternation, acquisition and reversal of a spatial discrimination in a T-maze. In each of these tasks, performance of 6-OHDA lesioned mice was improved relative to controls. Neurochemical analysis revealed that 6-OHDA lesioned mice exhibited a significant increase in the rate of sodium-dependent high affinity choline uptake in the hippocampus. These results are discussed in relation to current theories concerning the role of the septo-hippocampal complex and cholinergic system in the control of behaviour.

[Improvement in spontaneous and acquired spatial behaviors following lesions of septal dopaminergic afferents in mice: possible relations with hippocampal cholinergic activity]. / Amélioration de conduites spatiales spontanées et acquises après lésion des afférences dopaminergiques septales chez la Souris: relations possibles avec l'activité cholinergique hippocampique.

Recent evidence from pharmacological studies support the view that dopaminergic afferents to the septal complex which originate from the mesencephalic A10 area, exert a tonic inhibitory control over the activity of the septal-hippocampal cholinergic neurons. Accordingly one could predict that the release from such an inhibition by lesion of the septal dopaminergic terminals might improve performance in tasks known to be related to hippocampal cholinergic activity. In order to test this hypothesis mice of the C57BL/6 strain received a bilateral injection of 6-hydroxydopamine in the lateral septal nucleus; they were compared to subjects receiving saline and to unoperated control mice in tests performed in a T-maze: spontaneous alternation, acquisition and reversal of spatial discrimination. In all tasks, performance of experimental subjects was improved relative to controls. However, subsequent experiments showed that this improvement was not observed when visual (light/dark) discrimination was used. Finally, 6-hydroxydopamine injected mice exhibited a substantial increase in hippocampal sodium-dependent high affinity choline uptake (+ 16.7%). These results are discussed in relation to the three main theories concerning the role of the septo-hippocampal complex and cholinergic system in the control of behavior (i.e. Pavlovian internal inhibition, spatial mapping and working memory). Only the theory of spatial cognition seems to account for our present findings.

Facilitation of memory consolidation by post-training electrical stimulation of the medial septal nucleus: is it mediated by changes in rhythmic slow activity?

Sinusoidal (100 Hz) electrical stimulation was applied at a weak intensity (7.5 muA peak to peak) through bipolar electrodes located in the medial septal nucleus after partial acquisition of an appetitive operant conditioning task in a Skinner box. Analysis of performance in a retention test 24 hr later showed that (i) the presence of stimulation electrodes by itself impaired retention-test performance, and (ii) electrical stimulation applied 30 sec after the end of the acquisition session improves retention; this facilitatory effect disappeared when the treatment was delayed 15 min. Both impairment and facilitation were found to vary (considerably) among subjects. Electrodes located in the center of the medial septal nucleus led to both a greater impairment in unstimulated subjects and a greater facilitation in stimulated subjects than more anterior placements in the vicinity of the diagonal band. Finally, spectral analysis of hippocampal EEG showed that stimulation had no effect on rhythmic slow activity (RSA). These results are discussed in relation to studies showing that RSA is associated with memory-storage processes and our own hypothesis which underlines the importance of activation of septo-hippocampal cholinergic neurons in the early stages of these mnemonic processes.