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Cholinergic and GABAergic neurons in the medial septum-diagonal band of Broca (MS-DB) project to the hippocampus where they are involved in generating theta rhythmicity. So far, the functional properties of neurochemically identified MS-DB neurons are not fully characterized. In this study, MS-DB neurons recorded in urethane anesthetized rats and in unanesthetized restrained rats were labeled with neurobiotin and processed for immunohistochemistry against glutamic acid decarboxylase (GAD), parvalbumin (PV), and choline acetyltransferase (ChAT). The majority of the 90 labeled neurons (75.5%) were GAD+. Among them, 34.0% were also PV+, but none were ChAT+. Only 8.8% of the labeled neurons were found ChAT+. Remaining neurons (15.5%) were not identified. In anesthetized rats, all of the PV/GAD+ and 65% of GAD+ neurons exhibited burst-firing activity at the theta frequency. PV/GAD+ neurons displayed higher discharge rate and longer burst duration compared with GAD+ neurons. At variance, all of the ChAT+ neurons were slow-firing. Cluster-firing and tonic-firing were observed in GAD+ and unidentified neurons. In unanesthetized rats, during wakefulness or rapid eye movement sleep with hippocampal theta, the bursting neurons were PV/GAD+ or GAD+, whereas all of the ChAT+ neurons were slow-firing. Across the sleep-wake cycle, the GABAergic component of the septohippocampal pathway was always more active than the cholinergic one. The fact that cholinergic MS-DB neurons do not display theta-related bursting or tonic activity but have a very low firing rate questions how acetylcholine exerts its activating role in the septohippocampal system.

A chronic treatment with a cholinesterase inhibitor, donepezil (0.085 mg/kg/h for 30 days) increases significantly the number and amplitude of growth hormone (GH) pulses in 3- and 24-month-old rats without modifying nadir GH values. This treatment does not reduce age-related alterations in sleep/wake cycle but it increases immobility-related high-voltage spindles (HVS) in old animals. These data suggest that cholinergic mechanisms involved in age-related alterations in GH and sleep regulation are different.

GABAergic septohippocampal neurons play a major role in the generation of hippocampal theta rhythm, but modulatory factors intervening in this function are poorly documented. The neuropeptide somatostatin (SST) may be one of these factors, because nearly all hippocampal GABAergic neurons projecting to the medial septum/diagonal band of Broca (MS-DB) express SST. In this study, we took advantage of the high and selective expression of the SST receptor sst2A in MS-DB to examine its possible role on theta-related activity. Immunohistochemical experiments demonstrated that sst2A receptors were selectively targeted to the somatodendritic domain of neurons expressing the GABAergic marker GAD67 but were not expressed by cholinergic neurons. In addition, a subpopulation of GABAergic septohippocampal projecting neurons expressing parvalbumin (PV) also displayed sst2A receptors. Using in vivo juxtacellular recording and labeling with neurobiotin, we showed that a number of bursting and nonbursting neurons exhibiting high discharge rates and brief spikes were immunoreactive for PV or GAD67 and expressed the sst2A receptor. Microiontophoresis applications of SST and the sst2A agonist octreotide (OCT) showed that sst2A receptor activation decreased the discharge rate of both nonbursting and bursting MS-DB neurons and lessened the rhythmic activity of the latter. Finally, intraseptal injections of OCT and SST in freely moving rats reduced the power of hippocampal EEG in the theta band. Together, these in vivo experiments suggest that SST action on MS-DB GABAergic neurons, through sst2A receptors, represents an important modulatory mechanism in the control of theta activity.

Relationships between age-related changes in sleep patterns and neuronal activity have received scant attention. In the present study, reticularis pontis oralis (RPO) and ventral tegmental nucleus of Gudden (VTN) neurons were recorded in unanesthetized restrained young (3 months) and old (23 months) Sprague-Dawley rats during wakefulness (W), slow wave sleep (SWS) and rapid eye movement (REM) sleep. All RPO neurons displayed a tonic activity. Firing rates were similar during W in young and old rats. In contrast, firing rates were higher during SWS in old rats (P < 0.001). In both young and old rats, firing rates increased significantly during REM sleep as compared to W and SWS but this increase was markedly greater in old rats. Neurons recorded from VTN displayed bursting activity at theta frequencies during W and REM sleep. The frequency of VTN bursting neurons was higher during REM sleep as compared to W in both groups of age. This difference was significantly more pronounced in old as compared to young rats (P < 0.001). Sleep-related hyperactivity of pontine neurons is discussed in terms of a possible deficit in inhibitory processes in old rats.

Ghrelin, an endogenous ligand for the GHS receptor, stimulates GH secretion and gastrointestinal motility and has orexigenic effects. In this study, the relationships between ghrelin, GH secretion, feeding behavior, and sleep-wake patterns were investigated in adult male rats. The half-life of exogenous ghrelin (10 microg i.v.) in plasma was about 30 min. Repeated administration of ghrelin at 3- to 4-h intervals (one during lights-on and two during lights-off periods) increased GH release and feeding activity, and decreased rapid eye movement sleep duration. Endogenous plasma ghrelin levels exhibited pulsatile variations that were smaller and less regular compared with those of GH. No significant correlation between GH and ghrelin circulating levels was found, although mean interpeak intervals and pulse frequencies were close for the two hormones. In contrast, ghrelin pulse variations were correlated with food intake episodes in the lights off period, and plasma ghrelin concentrations decreased by 26% in the 20 min following the end of the food intake periods. A positive correlation between ghrelin levels and active wake was found during the first 3 h of the dark period only. In conclusion, ghrelin, in addition to affecting GH secretion, gastrointestinal motility, and feeding activity, also modifies sleep-wake patterns. However, a direct action of ghrelin per se or the indirect effects of feeding (and all of its attendant metabolic sequelae) on sleep cannot be differentiated. Moreover, ghrelin secretion is pulsatile and directly related to feeding behavior only.

It is well-established that rhythmically bursting (RB) activity in the medial septum is crucial for the generation of the hippocampal theta rhythm, but the contribution of other diencephalic-pontine structures is less documented. The ventral tegmental nucleus (VTn) of Gudden is related to the Papez's circuit via its interconnections with the medial mammillary nucleus, and therefore it may play a role in the generation of hippocampal theta. In the present study, extracellular activity from VTn neurons were recorded in unanesthetized restrained rats (n = 9). Hippocampal activity (EEG) and electromyograms were recorded simultaneously to identify sleep-waking states. RB activity was observed in VTn during wakefulness, with periods of hippocampal theta and during rapid eye movement (REM) sleep. Rhythmicity in VTn preceded theta activity in hippocampus. The frequency of RB neurons in VTn was 5.6 Hz during wakefulness and 6.8 Hz during REM sleep. It was similar to that of hippocampal theta. The rhythmicity was particularly stable and the firing rates were strikingly high during REM sleep. RB activity in VTn was also recorded from urethane-anesthetized rates (n = 3). Rhythmic firing (4.0 Hz) was slower than in unanesthetized rats and matched the urethane-related theta frequency. Our results show that neurons in VTn exhibit a marked RB activity during states of vigilance accompanied by hippocampal theta rhythm. They suggest that VTn may be a pontine hippocampal theta generator.

The effects of aging on the firing of septohippocampal neurons were estimated in unanesthetized, restrained young, old and very old rats (respectively 3, 23 and 30 months). Extracellular recordings were obtained during various states of arousal. The mean spontaneous activity for the overall neuronal population was not modified by aging. In contrast, the percentage of rhythmically bursting neurons was significantly lower in aged rats. During wakefulness, decrease of bursting activity was observed in old and very old rats (P<0.01 and P<0.001) whereas during rapid eye movement sleep it appeared only in the oldest group (P<0.01). The frequency of the bursts decreased in 30-month-old rats during wakefulness while it remained unchanged in both aged groups during rapid eye movement sleep. In old rats, at a time when the cholinergic septal neurons already deteriorated, a third of neurons recorded during rapid eye movement sleep exhibited a pattern of activity composed of long duration bursts with higher intraburst frequency than in young or very old rats. Our study shows that rhythmically bursting septal activity is impaired in aged rats and that the amplitude of the changes depends on advancing age and on states of arousal. Our findings suggest that age-induced loss and atrophy of cholinergic septal neurons contribute to the disorganization of the rhythmic activity but that functional alterations, influenced by the states of arousal, may also be considered.

To evaluate the influence of cholinergic projections from the basal forebrain on brain metabolism, we measured the cerebral metabolic rate of glucose (CMR(glu)) after unilateral lesioning of cholinergic basal forebrain neurons with the immunotoxin 192 IgG-saporin. CMR(glu) was determined in 24 cortical and 13 sub-cortical regions using the [14C]2-deoxy-D-glucose technique of Sokoloff. Average hemispheric CMR(glu) decreased by 7% (P<0.02) and 5% (P<0.05), 7 and 21 days after lesion, respectively. Regional effects were restricted to parietal and retrosplenial cortices, lateral habenula and the basal forebrain. We have previously shown that metrifonate increased CMR(glu) in intact rats. In lesioned rats, metrifonate (80 mg/kg, i. p.) was still active but the metabolic activation was reduced in terms of both the average hemispheric CMR(glu) and the number of regions significantly affected. Although it is reduced, the sustained effect of metrifonate in lesioned rats makes an argument for the use of this compound as treatment of cholinergic deficit in Alzheimer's disease.

The effects of subchronic administration of metrifonate, a long-lasting cholinesterase inhibitor, on local cerebral glucose utilization were assessed in 3- and 27-month old Sprague-Dawley rats, using the autoradiographic [14C]2-deoxyglucose technique. Rats were treated twice daily with metrifonate (80 or 120 mg/kg) for 3 weeks. The [14C]2-deoxyglucose experiment was performed 18 h after the last metrifonate administration. In 3-month old rats, metrifonate 80 mg/kg increased the average hemispheric cerebral glucose utilization by 12% (P > 0.001). Significant effects were observed in 19 of the 54 regions studied, including cortical and limbic regions. The higher dose induced a larger effect (average increase 17%, 24 of the 54 regions affected). In 27-month old rats, very similar effects were obtained. These results show that repeated administration of metrifonate leads to a sustained metabolic activation in rat brain, at a level comparable to the activation observed previously after a single administration of the drug.

Deficits in cholinergic function have been documented in a variety of brain disorders including Alzheimer's Disease and, to a lesser extent, in normal ageing. In the present article, we have reviewed our recent findings on the effects of the loss of basal forebrain cholinergic neurons on the functional properties of the septohippocampal pathway. In vivo and ex vivo investigations were performed in rats following basal forebrain cholinergic lesion with the specific immunotoxin 192 IgG-saporin. Our results suggest a significant contribution of cholinergic neurons in the rhythmically bursting activity recorded within the medial septum. In addition, they give evidence that acetylcholine may tonically decrease the glutamatergic synaptic responses in the hippocampus whereas the GABAergic mediated inhibitory potentials are not affected. The possible contribution of these cholinergic mechanisms in the age-related functional alterations of the septohippocampal activity is discussed.

The medial septum contains cholinergic and GABAergic neurons that project to the hippocampal formation. A significant proportion of the septohippocampal neurons (SHN) exhibit a rhythmically bursting (RB) activity that is involved in the generation of the hippocampal theta rhythm. The neurochemical nature of septal RB neurons is not firmly established. To address this question, the septal unit activity has been recorded in rats after selective destruction of the cholinergic septal neurons by the immunotoxin 192 IgG-saporin. Experiments have been performed in urethan-anesthetized and unanesthetized rats, 14-21 days after lesion. Acetylcholinesterase (AChE) histochemistry revealed a near-complete loss of cholinergic septal neurons and of cholinergic fibers in the hippocampus. The recorded neurons were located in the medial septum-diagonal band of Broca area. A number of these neurons were identified as projecting to the hippocampus (SHN) by their antidromic response to the electrical stimulation of the fimbria-fornix. In urethan-anesthetized lesioned rats, the percentage of RB neurons decreased significantly as compared with controls (17 vs. 41% for SHNs and 5 vs. 19% for unidentified septal neurons). The axonal conduction velocity and the burst frequency of the SHNs that retained a RB activity were higher in lesioned as compared with control rats. The number of spikes per burst was lower and the burst duration was shorter in lesioned rats as compared with controls. The urethan-resistant hippocampal theta was altered both in terms of frequency and amplitude. In unanesthetized lesioned rats, no RB septal neurons were found during arousal, as compared with 25% in controls. Their number was also markedly reduced during paradoxical sleep (9.7 vs. 38.5%). Histochemistry in 192 IgG-saporin-treated rats showed that RB neurons were found in areas devoid of AChE-positive neurons but containing parvalbumine-positive (presumably GABAergic) neurons. These data show that RB activity is considerably reduced after selective lesion of the cholinergic medial septal neurons. They suggested that the large majority of the RB septal neurons are cholinergic and that the few neurons that display RB activity in lesioned rats are GABAergic.

The effects of the centrally acting anti-cholinesterase metrifonate (MFT) and its metabolite dichlorvos (2,2-dichlorovinyl dimethyl phosphate; DDVP) on local cerebral glucose utilization (LCGU) have been studied in 3- and 27-month-old rats, using the autoradiographic [14C]deoxyglucose technique. In 3-month-old rats, MFT (80 mg/kg i.p.) increased LCGU significantly in 17 of the 54 regions studied, including insular, cingulate, and temporal cortices, ventral hippocampus, thalamus, lateral habenula, substantia nigra, and superior colliculus. In these regions, the average MFT-induced increase in LCGU was 23% above control. The average hemispheric LCGU increased by 10% (p < 0.01). DDVP (5 mg/kg) increased LCGU in 19 regions (average increase 26%). The average hemispheric LCGU increased by 9% (p < 0.01). Regional distributions of MFT- and DDVP-induced increases in LCGU were similar and overlapped the distribution of the acetylcholinesterase activity. In 27-month-old rats, MFT was active in 18 regions (average increase 25%). The whole-brain mean LCGU increased by 10% (p < 0.01). MFT compensated for the age-related hypometabolism in some brain areas including insular, temporal, and retrosplenial cortices, substantia nigra, and superior colliculus. The effects of MFT on LCGU were preserved in old rats, at variance with other anticholinesterases (tacrine, physostigmine). Which are less active in the aged rat brain.

The effects of the centrally acting anti-cholinesterases tacrine (tetrahydroaminoacridine, THA) and physostigmine (PHY), on local cerebral glucose utilization (LCGU) have been studied in 27-month-old rats, using the autoradiographic [14C]deoxyglucose technique. THA (10 mg/kg i.p.) increased LCGU significantly in 13 of the 54 regions studied (24%) including insular, parietal, temporal, and retrosplenial cortices, septohippocampal system, thalamus, lateral habenula, and superior colliculus. In these regions, the average THA-induced increase in LCGU was 24% above control. The whole brain mean LCGU was not significantly increased. PHY (0.5 mg/kg) increased LCGU in 18% of the regions (average elevation, 23%). The whole brain mean LCGU increased by 7% (p < 0.05). The regional distributions of THA- and PHY-induced increases in LCGU were extremely similar and overlapped the distribution of the M2 muscarinic receptors and that of acetylcholinesterase activity, suggesting that the major effects of THA and PHY on LCGU result from their anticholinesterase action. As compared to those of 3-month-old rats, both the number of regions affected and the amplitude of the metabolic activation were significantly less in aged rats. However, the drugs were still active in old rats and compensated for the age-related hypometabolism in some brain areas.

Intracerebroventricular injection of the toxin 192 IgG-saporin (4 micrograms) kills the cholinergic neurons of the basal forebrain bearing the low affinity NGF receptor (NGFr). The effect of this cholinergic denervation on the hippocampal and cortical electrical activity (EEG) was studied during sleep and wakefulness. EEG was recorded under freely-moving conditions in lesioned (n = 10) and control (n = 6) rats (8-16 days post-injection). In lesioned rats, active (AW) and quiet (QW) wakefulness episode durations were similar to those of controls whereas the REM sleep duration was reduced, 8 days post-lesion (P < 0.01). Bouts of REM sleep were more numerous but shorter. The hippocampal theta activity was still present in lesioned-rats during AW (type 1 theta), QW (type 2 theta) and REM sleep. The frequency was unchanged but the amplitude of the three types of theta was significantly reduced (P < 0.01). Type 2 theta occurred with shorter and less regular bouts (P < 0.05). Abnormal slow waves (2-4 Hz) were observed during wakefulness. Histology showed a dramatic loss of NGFr-positive neurons in the basal forebrain and a decline in hippocampal and cortical acetylcholinesterase activity. These results suggest that the cholinergic septohippocampal input is not the primary pacemaker for the hippocampal theta rhythm.

Human amnesia cases (after surgical removal of the hippocampi or brain anoxia) have clearly established the critical role of the hippocampal formation in anterograde amnesia. Other parts of the brain may also contribute to anterograde amnesia (mammillary bodies, medial thalamus). In neurodegenerative diseases (and specially in Alzheimer's disease) amnesia is often the prominent symptom, but the brain lesions are not restricted to the hippocampal formation. In Alzheimer's disease they involve also the cerebral cortex and several subcortical nuclei. Physiological brain aging is also associated with some degree of memory impairment, but much less severe than in Alzheimer's disease. The issue of the nature and the mechanisms of the memory impairment associated with age and with Alzheimer's disease is very important, because the frequency of these problems increases dramatically as the populations of the world is growing older. There is some evidence that neuronal loss and alterations in neurotransmitter systems occur in the aged subject, but the relationship between such changes and the age-related memory deficit is far from being clear. In Alzheimer's disease, the loss of memory is likely to be due to neuronal loss in cerebral cortex and hippocampal formation, along with alterations in neurotransmitter systems (specially cholinergic, monoaminergic and aminoacidergic systems). The work in experimental animals has largely confirmed the critical role of the hippocampal formation, as well as identified other critical structures. The mechanisms of the age-related memory impairment can be to some extent investigated in aged animals. In the aged rat there is evidence that several neurotransmitter networks are altered. Alteration in the dopaminergic and cholinergic systems have been extensively studied, but the involvement of other systems is likely. Learning and memory deficits are consistently observed in a sub-population of aged rodents (as well as in other species including non-human primates). For instance some aged rats do have a deficit in the performance of a spatial learning task such as the "water maze". There is some evidence that this deficit is due, at least in part, to alterations in the functions of the hippocampal formation. In other words, if aged rats have a spatial memory deficit, it might be due to changes in hippocampal neuronal circuitry. The study of age-related alterations in hippocampal neuronal networks, using electrophysiological techniques have shown that several neuronal properties such as resting membrane potential, membrane resistance or sodium spike amplitude are not altered in the aged rat hippocampus.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of the anticholinesterases tetrahydroaminoacridine (THA) and physostigmine on local cerebral glucose utilization (LCGU) were studied in the conscious rat, using the autoradiographic [14C]deoxyglucose technique. THA (5 mg/kg i.p.) increased LCGU significantly in 8 of the 43 regions studied. A higher dose of THA (10 mg/kg) produced a metabolic activation in 19 of the 43 regions. LCGU increased in cortical areas (including parietal and temporal cortices), the septohippocampal system, the thalamus, the lateral habenula, the basolateral amygdala, the superior colliculus, and the substantia nigra. Scopolamine (4 mg/kg i.p.) reversed the THA-induced LCGU increase. Physostigmine (0.2 and 0.5 mg/kg) increased LCGU in 15 and 22 regions, respectively. The average magnitude of the change induced by 0.5 mg/kg of physostigmine was similar to that observed after THA at 10 mg/kg, but the topography of the effects was somewhat different. Physostigmine increased LCGU in the preoptic magnocellular area, the brainstem, and the cerebellum but not in the parietal cortex. The effects in the septohippocampal system were smaller than those induced by THA. The regional topography of the LCGU increase overlapped the distribution of the M2 muscarinic receptors and that of acetylcholinesterase activity. These data suggest that the major effects of THA and physostigmine on LCGU result from their anticholinesterase action.

Unilateral nucleus basalis magnocellularis (NBM) ablation, which causes partial cholinergic denervation of the ipsilateral anterior neocortex, results in an acute but transient depression of regional cerebral metabolic rates for glucose (rCMRglc) in deafferented areas; rCMRglc normalizes within 2 weeks. To seek possible compensatory changes in cholinergic mechanisms following NBM ablation that could lead to rapid metabolic normalization, we studied rCMRglc responses to the receptor agonists nicotine and arecoline and the cholinesterase inhibitor physostigmine in rats at 2 weeks after unilateral NBM destruction. Physostigmine increased rCMRglc in 10 of 30 cortical areas contralateral to the NBM lesion. Compared to the unlesioned side, rCMRglc after physostigmine in the lesioned cortex was significantly lower in 2, significantly higher in 1 and not different (P < 0.05) in 27 areas. Neither arecoline nor nicotine treatment produced rCMRglc asymmetry in lesioned rats. These results demonstrate that responsivity to physostigmine is maintained in most regions of the rat neocortex after extrinsic cholinergic denervation by NBM ablation. This adaptive response appears not to result from cholinergic receptor upregulation and may reflect instead reorganization of cholinergic synapses.

We have performed a qualitative and quantitative analysis of the electrophysiological properties of medial septal neurons in the unanesthetized rat. The rat's head was held in a stereotaxic apparatus by a painless head-restrained system that was implanted seven days prior to the recording sessions. Extracellular recordings were made in a mixed population of antidromically identified septohippocampal neurons and unidentified medial septal neurons in different states of arousal and in response to peripheral and reticular stimulations. The spontaneous activity as well as the percentage of rhythmically bursting septal neurons varied significantly according to the state of arousal. Higher values were noted in paradoxical sleep (28 imp/s and 94% of bursting neurons) as compared with wakefulness with hippocampal theta rhythm (17.4 imp/s and 64.2% of bursting neurons) and slow wave sleep (12.3 imp/s and 8% of bursting neurons). The frequency of the bursts was significantly higher during paradoxical sleep. In individual medial septal neurons, arousing stimuli and paradoxical sleep could induce rhythmic bursting activity in previously non-bursting neurons provided that they were fast-firing neurons. No differences were noted in the functional characteristics of neurons in the medial septal nucleus as compared with the diagonal band of Broca. When the unanesthetized rats were compared with a group of urethane-anesthetized rats, the spontaneous activity was higher and more irregular in the absence of anesthesia. The percentage of the bursting neurons was significantly lower in the unanesthetized rats (32.3% vs 43.3%). However, the frequency of the bursts was higher (5.9 +/- 0.1 Hz vs 3.5 +/- 0.1 Hz). Since the patterns of activity of medial septal neurons fluctuate in different physiologically relevant states, previous classifications of these neurons made by ourselves and other authors, in urethane-anesthetized rats, may not be appropriate.

Tianeptine is a tricyclic antidepressant which enhances serotonin uptake in certain brain areas. Tianeptine has been reported to improve both working and reference memories in rodents. The effects of tianeptine on the spontaneous activity of medial septal neurons were studied in rats anesthetized with urethane. Systemic administrations (0.2-1 mg/kg i.v.) of tianeptine decreased the spontaneous activity and disorganized or suppressed the rhythmically bursting activity of medial septal neurons, in a dose related manner. Iontophoretic administrations of tianeptine did not modify the spontaneous activity of medial septal neurons. Changes of the bursting activity were inconsistent. However, tianeptine blocked partially or completely the inhibition induced by the serotonin in 68% of the cases. In contrast, other antidepressants (amitriptyline, clomipramine and fluoxetine) potentiated the inhibitory effect of serotonin in 50%-60% of the cases. Our results show that tianeptine, applied by iontophoresis, has an effect on the medial septal neurons which was opposite to that of other antidepressants. On the basis of our findings, it can be tentatively proposed that tianeptine may have a beneficial effect on memory by counteracting the serotonin-induced inhibition of medial septal neurons.

The effects of the iontophoretic applications of glutamate (Glu), gamma-aminobutyric acid (GABA), acetylcholine (ACh) and carbachol (CARB) were studied on neurons located in the medium septal area (MSA) in the unanesthetized rat. In the absence of anesthesia, functional properties of the MSA neurons were significantly different from those observed in the urethane-anesthetized rat (higher variability of discharge rate, lower percentage of rhythmically bursting neurons). Glu excited 80% and GABA inhibited 96% of the MSA neurons. These percentages were similar to those obtained in the urethane-anesthetized rats. In contrast, the percentage of neurons excited by ACh (28%) or by CARB (27.2%) were significantly lower than in the urethane-anesthetized rat. Our results suggest that urethane might alter cholinergic sensitivity in the MSA and confirm that anesthesia can induce a bias in the iontophoretic study of some brain structures.

Age-related changes in the levels of dopamine (DA) and its metabolites were measured in seven cerebral cortical areas and in the striatum of 3, 10 and 27 month-old Sprague-Dawley rats. An age-related increase in DA levels was observed in the somatomotor (SM) cortex. In contrast, a decrease was observed in the temporal (T) cortex. Decreases in homovanillic acid (HVA) levels were observed in prelimbic (PL), pyriform (PY) and T cortex of aged rats, whereas significant increases in the levels of 3-methoxytyramine (3-MT) were observed in PL, prefrontal (PF), cingulate (C) as well as in T cortex. In the striatum, DA and HVA were decreased but the level of 3-MT remained unchanged. Norepinephrine (NE) levels increased in rats from 3 to 27 months in all the cortical areas. The increase in the levels of the DA extraneuronal metabolite, 3-MT, confirms our previous results showing that the release of DA might be increased with age in some cortical areas. The present results show that there is no general age-related decrease in the level of monoamines and of their metabolites in the rat cerebral cortex and that the changes display a complex, area-specific pattern.

The effects of tetrahydro-9-aminoacridine (THA), an anticholinesterase drug, have been studied in the rat both in vivo (cerebral cortex) and in vitro (CA1 field of the hippocampus) and compared with those of physostigmine. In the cerebral cortex THA potentiated the excitatory effect of acetylcholine in most neurons, including cortical neurons recorded from chronic unanesthetized animals. In vitro, THA (but not physostigmine) had a depolarizing, atropine- and tetrodotoxin-insensitive effect. This effect is associated with an increase in membrane resistance which suggests a direct effect of THA on hippocampal neurons. In addition THA blocked the slow inhibitory postsynaptic potential. At the same concentration THA potentiated the slow cholinergic excitatory postsynaptic potential produced by electrical stimulation of the cholinergic afferents. Its potency was, however, about 10 times lower than that of physostigmine. These results show that THA: (1) is an anticholinesterase much less potent than physostigmine; but (2) has also direct effects on central neurons, not observed with physostigmine and unrelated to its anticholinesterase activity.

Dihydroxyphenylalanine (DOPA) and its metabolite 3-O-methyl-DOPA (3-OMDOPA) have been identified as normal constituents in blood, CSF and brain in human and several animal species. In the present study, we report results of 3-OMDOPA measurements in seven cortical areas, hippocampus and striatum of young (3-month-old), middle aged (10-month-old) and aged (27-month-old) rats. 3-OMDOPA was identified in all the areas considered. Regional differences were observed, with the highest levels being found in the hippocampus and the somatomotor and pyriform cortex. A marked increase in the level of 3-OMDOPA was observed in all the cortical areas and in the hippocampus of aged rats. In contrast, this level remained unchanged in the striatum. These modifications were compared to those observed in the levels of dopamine (DA) in the same areas.

Electrophysiological approaches range from global EEG studies to single unit recordings. Quantified EEG studies indicate that cognition enhancers increase the power of fast-frequency (beta) EEG activity in humans and the duration of hippocampal theta in animals. A problem here is the distinction between improved cognition and increases in general arousal, although the two might have the same end effect. A second global approach is the use of the evoked potential or even the conditioned evoked potential, for example the P 300 wave. This approach, using defined brain circuits, would appear to be more specific than global EEG models. Both approaches can be used in parallel studies in animals and man. Studies at more cellular levels can only be used in animals and suffer from severe constraints. The most precise approach, single cell recording, cannot be conducted in freely behaving animals, whereas multi-unit approaches in awake animals tend to produce many artefacts. However, techniques using microintophoretically applied drugs in freely moving rodents and monkeys can contribute to the understanding of mechanisms of action. In vitro studies, for example long-term potentiation in hippocampal slices, may represent a neuronal model of learning but its role in normal brain function has still not been elucidated.

The response of somatosensory cortical neurons to iontophoretic applications of monoamines was studied in unanesthetized rats. The animal's head was held in a stereotaxic apparatus by means of a painless head-restraining system implanted 8 days prior to the recording sessions. The electrodes consisted of a recording micropipette attached to a multibarreled iontophoresis micropipette. The electrode penetrations were reconstructed on camera lucida drawings of frontal brain sections. The percentage of cortical neurons responding to application of monoamines were 76% after noradrenaline, 58% after dopamine and 66% after serotonin. The differences observed among percentages of responses seemingly correlate with the relative abundance of terminal axons and receptors for each of the three monoamines in the somatosensory cortex. The vast majority of the responding neurons were inhibited by monoamines and this inhibitory effect was independent of the level of spontaneous activity. The depressant effect of the monoamines on glutamate and acetylcholine-evoked responses supports a modulatory role for these substances. Serotonin was the most potent, followed by noradrenaline and dopamine. The present study shows that when the influence of anesthesia is eliminated, the predominant effect of monoamines on cortical first somatosensory neurons is one of inhibition. These findings contrast with results obtained under some anesthetic conditions, as well as under in vitro conditions.

Experiments have been performed on unanesthetized rats using a chronic restraint system. The animal's head was held in a stereotaxic apparatus by means of two metallic tubes fixed on the skull with dental cement. Electrodes consisted of a recording micropipette (filled with 1 M NaCl and 2% Pontamine Blue) attached to a multibarreled micropipette for iontophoresis. Electrode penetrations were reconstructed on camera lucida drawings of frontal brain sections. The overall percentage of spontaneously active somatosensory neurons was 77% with a mean spontaneous activity of 5.9 impulse/s (n = 405). Yet differences were observed in the proportions of active cells as well as in the mean spontaneous activity between cortical layers (both parameters being significantly higher in layers V and VI). Comparison with results obtained under urethane anesthesia [Dykes R. W. and Lamour Y. (1988) J. Neurophysiol. 60, 703-724] shows that the percentage of the spontaneously active neurons and the mean spontaneous activity were both significantly higher in unanesthetized rats (77 vs 36%; 5.9 vs 2.6 impulse/s). Nevertheless, the laminar distribution of the most active cells was similar under both conditions. In the present study, 52.3% of the neurons (n = 380) were excited by acetylcholine and 46% (n = 198) by carbachol. Significantly larger percentages of neurons excited by acetylcholine were found in layers Vb and VIb. These effects of cholinergic agonists--observed for the first time in unanesthetized rats--differed significantly from those previously obtained under anesthesia (33 and 34% of neurons excited by acetylcholine and carbachol, respectively) [Lamour Y. et al. (1982) Neuroscience 7, 1483-1494].(ABSTRACT TRUNCATED AT 250 WORDS)

Cortical and thalamic EEG and somatosensory evoked potential (SEP) induced by stimulation of the somesthetic radiations were studied in scrapie-infected rats. Animals were inoculated intracerebrally with a rat-adapted strain (originating in the C506 M3 mouse scrapie strain). EEG and SEP were recorded from 9 to 17 months after inoculation (ti). Abnormalities (paroxysmal bursts, isolated spikes) first occurred in the cortex (parietal areas) and later in the thalamus, where they were usually less marked. Latencies of the postsynaptic components of the SEP increased at ti + 9 months. This effect became progressively more pronounced and at ti + 15 months, latencies of presynaptic components were also delayed. Nevertheless, marked alteration of the SEP occurred only at the terminal stage of the disease. These findings show that the scrapie-induced disturbances affect more especially the cortex. Decrease of inhibitory processes as well as electronic coupling between cells, resulting from the virus-induced membrane fusion, could produce paroxysmal activity of EEG and SEP impairments.

The effects of various psychotropic drugs (benzodiazepines, antidepressants, neuroleptics and nootropic drugs, a family of cognition activator agents) on firing rates of septohippocampal neurons, identified by electrical antidromic stimulation, were studied in the medial septum-nucleus of the diagonal band of Broca of rats anaesthetized with urethan. Extracellular potentials from single septohippocampal neurons were recorded using glass pipettes. Drugs were applied by either microiontophoresis or intravenous injections (i.v.). Benzodiazepines produced a marked depression of spontaneous firing rates of septohippocampal neurons whether applied i.v. (diazepam) or iontophoretically (flurazepam, midazolam). In addition, diazepam had a potent depressant effect on the rhythmically bursting activity of the septohippocampal neurons. Baclofen also had an inhibitory effect. Antidepressant drugs (applied by iontophoresis) as well as amphetamine, had a depressant effect on spontaneous firing rates. Neuroleptics (i.v.) had less significant or consistent effects on septohippocampal neurons, although the effects of haloperidol were usually inhibitory. Nootropic drugs were generally ineffective. These data indicate that most psychotropic drugs tested (with the exception of nootropic drugs) have an inhibitory effect on the spontaneous activity of septohippocampal neurons. However, benzodiazepines seem to be more active than antidepressants or neuroleptics. Oxotremorine (i.v.) had a potent excitatory effect on septohippocampal neurons. Atropine (i.v.) increased the septohippocampal neurons' firing rate in some cases. These results are discussed in view of the possible implication of the involvement of septohippocampal neurons in the mediation of the effects of psychotropic drugs on the central nervous system and, more specifically, on the cholinergic systems.

Age-related changes in the content of dopamine (DA), homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in anterior cerebral cortex, hippocampus and striatum of the rat have been investigated using HPLC with electrochemical detection. A significant decrease in HVA was observed in the striatum and hippocampus of the aged (27 months) animals, as compared to the controls (2.4 to 2.6 months). A significant decrease in DA levels was also observed in the hippocampus but not in the striatum. In contrast, the level of DA in the cerebral cortex was markedly increased in the aged animals. A concomitant increase in 3-MT level was observed. Finally the level of 5-HIAA was significantly increased in striatum and hippocampus.

Levels of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethyleneglycol (DHPG) were determined by high-performance liquid chromatography in different brain areas of scrapie-infected rats, 8.5 months after intracerebral inoculation of a rat-adapted strain from mice brain (C 506). At this time, rats developed early clinical signs of the disease. Scrapie-infected rats showed a reduction in the levels of 5-HT and 5-HIAA (frontal cortex, hippocampus, mesolimbic structure). Concentrations of DHPG decreased in the frontal and parietal cortices but remained unchanged in the hippocampus. DOPAC levels decreased in the striatum but not in the mesolimbic structure. These results confirm that the serotonergic, noradrenergic and dopaminergic systems are altered in the brain of scrapie-infected rats. They can partly account for clinical signs of scrapie and are in agreement with the scarce data provided by the postmortem analysis of Creutzfeldt-Jakob disease brains.

The properties of neurons of the medial septal nucleus and of the nucleus of the diagonal band of Broca which project to hippocampus or to interpeduncular nucleus were compared in rats anesthetized with urethane. Neurons projecting to the interpeduncular nucleus had a slower conduction velocity and a lower spontaneous discharge rate. In contrast, their responses to various putative neurotransmitters (glutamate, GABA, acetylcholine) were similar. In a few cases, neurons projecting to both structures (i.e., with branched axons) were observed. Both septohippocampal and septointerpeduncular pathways are known to be partly cholinergic. Our results show that they originate from two independent populations of medial septal-nucleus of the diagonal band of Broca neurons with different physiologic properties.

Invasion of scrapie agent into the central nervous system (CNS) was studied in rats following intracerebral and peripheral inoculation, the latter by injection into intact or transected sciatic nerve. Comparison of sleep-wakefulness alterations, neuropathological features, and time lag of electroencephalographic and clinical signs in the 3 groups suggests that hematogenous spread of infection to the CNS may predominate over neural transport, and that peripheral inoculation may closely approximate natural infection.

The medial septal area contains neurons which project to the hippocampal formation. A sizeable proportion of these septo-hippocampal neurons (SHNs) are cholinergic. About 40% of them also display a characteristic discharge pattern in rhythmic bursts. We hypothesized that SHNs with a rhythmically bursting activity (RBA) are the cholinergic ones. To test this hypothesis we studied the effects of acetylcholine synthesis blockade by hemicholinium-3 (HC-3) on the properties of the SHNs. HC-3 (16, 32 or 64 micrograms total dose) or saline were injected in the lateral ventricles of adult male Sprague-Dawley rats anesthetized with urethane. Extracellular recordings from SHNs in the medial septal area were obtained within hours after HC-3 injections (n = 24 animals). SHNs were identified by their antidromic response following electrical stimulation of the fimbria-fornix. The pharmacological properties of SHNs were studied in some animals using microiontophoretic applications from multibarreled electrodes filled with various neurotransmitters. The hippocampal rhythmic slow activity (RSA or theta) was abolished even after the lowest dose of HC-3 tested (16 micrograms). No significant change in SHNs conduction velocity or spontaneous activity was observed at any dose of HC-3. The percentage of SHNs with RBA was unchanged. In contrast the mean frequency of the RBA was decreased by HC-3 in a dose-dependent fashion. The mean frequency was lowest within the first 3 h after injection. Although the mean spontaneous activity was unchanged SHNs tended to have more spikes per burst. The effects of various neurotransmitters on SHNs were qualitatively unchanged after HC-3 injection. These results suggest that acetylcholine synthesis blockade by HC-3 leads not only to the disappearance of the hippocampal RSA in urethane-anesthetized animals, but also to a decrease in the frequency of the rhythmically bursting activity of the SHNs. Since the 4-Hz hippocampal theta is atropine-sensitive, the results provide indirect evidence that the SHNs with rhythmically bursting activity are the cholinergic SHNs.

Electrophysiological and pharmacological properties of neurons within solid basal forebrain transplants were studied in adult rats anesthetized with urethane. No specific topography of the neurons recorded was observed within the graft. The mean spontaneous activity of the grafted neurons (GNs) was relatively low (4.9 impulses/s) but not unlike that of other central neurons in situ. A large proportion of GNs fired with regular discharges, but other modes of discharge were also observed. A few rhythmically bursting GNs were recorded having a discharge pattern very much like that of the rhythmically bursting medial septal neurons. The responses of GNs to glutamate, gamma-aminobutyric acid, acetylcholine, serotonin and norepinephrine was fairly similar to those described in other central structures.

Scrapie was transmitted (1st passage) to various strains of rats through intracerebral (IC) inoculation of sheep-scrapie, mouse-scrapie and hamster-scrapie brains. For the 2nd passage, Wistar rats were chosen and inoculated IC with rat-scrapie brain from the 1st passage. Three groups of 12 rats were considered (S for sheep, M for mouse, H for hamster). Electrodes were chronically implanted for EEG recording. From 100 to 110 days after inoculation, EEG abnormalities were observed, consisting of spindle-shaped bursts of diphasic spikes sporadically occurring during quiet wakefulness. Between 130 and 230 days, EEG signs diversified and became more pronounced. Simultaneously, changes occurred in the sleep-wakefulness cycle: active wakefulness decreased and was replaced by quiet wakefulness (rats were motionless , as if prostrate ). Slow wave sleep diminished, giving way to an unstable low voltage sleep. EEG disturbances clearly preceded the onset of the clinical signs (the latter appearing at about the 8th month). The disease was fairly similar in the S, M and H groups. The interest of the experimental scrapie rat model is discussed in the framework of current research on the neurophysiology of the subacute spongiform encephalopathies.

The performances of aged (24-26 months) rats in two behavioral tasks (passive avoidance and spontaneous alternation) have been studied. Subpopulations of old animals were found to be impaired in these tasks. Most of the impaired animals, however, were not impaired in both tasks. The properties of the septo-hippocampal neurons (SHNs) were subsequently studied in the same group of experimental animals, anesthetized with urethane, using electrophysiological techniques. The spontaneous activity of SHNs displaying a rhythmically bursting activity (RBA) was significantly higher in animals impaired in the spontaneous alternation task. The proportion of SHNs with RBA was significantly lower and the frequency of the RBA was higher in animals impaired in the passive avoidance task. The pharmacological properties of the SHNs were not significantly different in the various groups. The significance of these complex correlations with regard to the age-related alterations of SHNs properties is discussed.

The effects of the scrapie agent on the levels of monoamines and their metabolites, and on choline acetyltransferase (CAT) activity have been investigated in discrete brain areas in the rat. Two strains of scrapie (8745 from sheep brain and C506 M3 from mice brain) were inoculated. Scrapie-infected rats showed a reduction in the levels of serotonin (prefrontal cortex, hippocampus, striatum) and dopamine (striatum) and an elevation of 5-HIAA levels (cerebral cortex, striatum, thalamus). Noradrenaline levels were decreased only in the cerebral cortex and cerebellum of rats infected with the scrapie strain C506 M3. CAT activity remained unchanged. These data suggest that the scrapie agent causes a derangement of noradrenergic, serotonergic and dopaminergic systems in the rat brain.

The spontaneous activity of CA1 pyramidal cells was reduced with microelectrodes from the hippocampus of curarized adult rabbits under painless fixation. A statistical analysis of the data was made by a computer, using a program developed to process time interval series. 1. Various temporal patterns of discharge were observed. A classification into 5 distinct patterns could be disclosed, based on statistical parameters, particularly expectation density, joint interval histogram and interspike interval histogram. 2. The recorded neurones were distributed unequally in these groups, the distribution varying with sleep and wakefulness. 3. Some firing patterns prevailed during wakefulness and some during sleep, but all of them were observed in both states and no one was considered specific to one state. 4. The variability in neuronal discharge was estimated. "Stable" cells (60%) exhibited only one firing pattern. "Unstable" cells (40%) exhibited either two (82%) or three (18%) types of firing. 5. 65% of the cells recorded during waking and then during sleep shifted their firing pattern. The majority of the other units (35%), which kept the same pattern of discharge, were already stable during waking. Hence, they exhibited only one firing pattern and did not appear to be affected by sleep or waking.

The septo-hippocampal neurons (SHNs), located in the medial septum, project to the hippocampal formation. The population of SHNs, as shown by single unit recordings in urethane-anesthetized rats, is heterogeneous, both in terms of patterns of spontaneous activity (a significant proportion of the SHNs display a characteristic rhythmically bursting activity at about 4 Hz) and of conduction velocity. Their average rate of spontaneous discharge is quite high (20 impulses per second). They are excited by the iontophoretic application of acetylcholine and various cholinergic agonists. They are also excited by some peptides such as substance P and TRH. Parallel studies in aged animals show that the physiological properties of the SHNs are altered, while their pharmacological properties seem to be unchanged. Immunohistochemical investigations using antibodies against various peptides and a monoclonal antibody against choline acetyltransferase (ChAT) show that SHNs retrogradely-labeled from the hippocampus often contain ChAT, less frequently galanin-like immunoreactivity and in a few cases enkephalin, luteinizing hormone-releasing hormone, or calcitonin gene-related peptide. In contrast, cholecystokinin, vasoactive intestinal peptide, substance P, somatostatin, dynorphin-B and neurotensin, although present in some medial septal neurons, were never observed in neurons projecting to the hippocampus.