Donepezil restores GH secretion in old rats without affecting the sleep/wake cycle.

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.

Sleep-related increase in activity of mesopontine neurons in old rats.

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.

Decrease in calbindin content significantly alters LTP but not NMDA receptor and calcium channel properties.

The contribution of the cytosolic calcium binding protein calbindin D(28K) (CaBP) to the synaptic plasticity was investigated in hippocampal CA1 area of wild-type and antisense transgenic CaBP-deficient mice. We showed that long-term potentiation (LTP) induced by tetanic stimulation in CaBP-deficient mice was impaired. The fundamental biophysical properties of NMDA receptors and their number were not modified in CaBP-deficient mice. We also demonstrated that the physiological properties of calcium channels were identical between genotypes. An insufficient Ca(2+) entry through NMDA receptors or calcium channels, or a decrease in NMDA receptor density are unlikely to explain this impairment of LTP. Interestingly, we showed that the loss of LTP was not prevented by glycine but was restored in the presence of a low concentration of the NMDA receptor antagonist D-APV (5 microM) and of the calcium chelator BAPTA-AM (5 microM). Moreover, we observed a loss of LTP in the wild-type mice when the postsynaptic tetanic-induced [Ca(2+)](i) rise is excessively increased. Conversely, a weaker tetanus stimulation allowed LTP induction and maintenance in CaBP-deficient mice. These results suggest that a higher cytosol [Ca(2+)](i), due to the decrease of CaBP expression may impair LTP induction and maintenance mechanisms without affecting the mechanisms of calcium entry. Thus, CaBP plays a critical role in long term synaptic plasticity by limiting the elevation of calcium rise in the cytosol to some appropriate spatio-temporal pattern.

Ventral tegmental nucleus of Gudden: a pontine hippocampal theta generator?

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.

NMDA receptor activation in the aged rat hippocampus.

Age-related alterations of N-methyl-D-aspartate receptor (NMDAr) activation were investigated in the CA1 field of hippocampal slices from young (3-6 months old) and aged (25-33 months old) Sprague-Dawley rats by using ex vivo extracellular electrophysiological recording techniques. NMDAr-mediated field excitatory postsynaptic potentials (fEPSPs) were induced by electrical stimulation of glutamatergic fibers in a magnesium (Mg(2+))-free medium supplemented with the non-NMDAr antagonist CNQX. The fEPSPs were significantly smaller in aged rats, whereas the response of presynaptic afferent fibers remained unaffected. No significant age-related differences were found in the ability of Mg(2+) to depress the magnitude of NMDAr-mediated fEPSPs. The responsiveness of postsynaptic NMDAr to the agonist was assessed in both groups of animals. No age-related differences were recorded either in the depolarizing effect of bath-applied NMDA or in the magnitude of the depolarization after altering extracellular Mg(2+) concentration. Finally, short-term potentiation (STP) of excitatory transmission was studied in young and aged rats considering the pivotal role of NMDAr in synaptic plasticity. No age-related alterations of the magnitude and the time course of STP in response to 10 or 30Hz conditioning stimulation were found. Because of the decrease in the magnitude of NMDAr-mediated synaptic transmission in aged animals, the absence of obvious modifications of synaptic plasticity suggests the occurrence of compensatory mechanisms that are discussed.

Age-related changes in rhythmically bursting activity in the medial septum of rats.

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.

Sustained effect of metrifonate on cerebral glucose metabolism after immunolesion of basal forebrain cholinergic neurons in rats.

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.

Loss of rhythmically bursting neurons in rat medial septum following selective lesion of septohippocampal cholinergic system.

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.

Effect of subchronic metrifonate treatment on cerebral glucose metabolism in young and aged rats.

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.

Immunolesion of the cholinergic basal forebrain: effects on functional properties of hippocampal and septal neurons.

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.

Behavioural pain-related disorders and contribution of the saphenous nerve in crush and chronic constriction injury of the rat sciatic nerve.

This study evaluated the pain-related behaviours induced by 2 models of peripheral sciatic nerve injuries in the rat: transient nerve crush and chronic constriction injury (CCI). Various lesions of the saphenous nerve were performed in order to investigate the role of saphenous innervation in behavioural disorders induced by these nerve injuries. Behavioural testing included assessment of responses to phasic stimulation (mechanical and thermal) and observation of 'spontaneous' pain-related behaviour. Results confirmed that the model of CCI induces marked and prolonged phasic and spontaneous pain-related disorders (up to week 7). Rats with crush injury exhibited moderate and transient hyperalgesia and allodynia to mechanical and thermal stimulation on the lesioned side (with a maximum at day 3 and a recovery by week 1). Section plus ligation of the ipsilateral saphenous nerve on the day of surgery prevented nociceptive behaviours and induced persistent mechanical and thermal anaesthesia or hypoesthesia of the lesioned paw in both models (lasting up to 3-4 weeks). Section without ligation of the saphenous nerve induced comparable results in rats with sciatic crush, but did not significantly modify nociceptive behaviours in rats with CCI. These data emphasise the role of adjacent saphenous nerve in the mechanisms of pain-related disorders induced by these peripheral nerve lesions. On the contralateral paw, pain-related modifications were also observed in both models, suggesting that unilateral nerve lesions induce remote modifications extending beyond the site of the injured nerve.

The age-related increase in galanin binding sites in the rat brain correlates with behavioral impairment.

The regional distribution of [125I]galanin specific binding sites was determined in young (three- to four-month-old), 14-15-month-old and aged (26-27-month-old) male Sprague-Dawley rats, previously tested for their performances in the Morris water-maze task, using the radioautographic method on brain sections. A significant increase in specific binding was observed in piriform and entorhinal cortex, ventral subiculum, and dorsal dentate gyrus in the aged rats, whereas no significant changes were observed in dorsal subiculum, amygdala, septal area and various subcortical structures. The area-specific regional increase in specific binding density in aged rats was significantly correlated with the impairment of the behavioral performance in the Morris water-maze task. The change in [125I]galanin specific binding was a result of an increase in the number of galanin binding sites, but not of an increase in affinity.

Tetrahydroaminoacridine and physostigmine increase cerebral glucose utilization in specific cortical and subcortical regions in the rat.

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.

Age-related increase in galanin-binding sites in the rat brain: correlation with behavioral impairment.

The regional distribution of 125I-galanin specific binding sites was determined by radioautography on brain sections in young (3- to 4-month-old) and aged (26- to 27-month-old) male Sprague-Dawley rats, previously tested for their performances in the Morris water maze task. In aged rats, a significant increase in specific binding was observed in piriform, perirhinal and entorhinal cortex, the CA1 field of the ventral hippocampus, ventral subiculum, and dorsal dentate gyrus, whereas no significant change was observed in the ventral dentate gyrus, the dorsal subiculum, the CA3 field of the hippocampus, the amygdala or the septal area. The area-specific regional increase in specific binding density in aged rats was significantly correlated with the impairment of their behavioral performances in the Morris water maze task. The change in 125I-galanin specific binding in the aged rats was a result of an increase in the number of galanin-binding sites, without change in affinity.

Time course of degeneration and regeneration of myelinated nerve fibres following chronic loose ligatures of the rat sciatic nerve: can nerve lesions be linked to the abnormal pain-related behaviours?

This study of a mononeuropathy of 1-15 weeks (W) duration was induced in rats by setting 4 loose ligatures around the common sciatic nerve. This chronic lesion, in which the continuity of the nerve was maintained, has been introduced as a model for experimental pain. Quantitative analyses of teased nerve fibres and a morphometric analysis of semi-thin transverse sections, were performed and completed by electron microscopic examination. Morphological changes were observed mainly distal, but also proximal, to the ligatures, indicating significant axonopathy with simultaneous degeneration and regeneration. The lesions were analysed in parallel with the time course of the pain-related behaviours. Both were at their maximum 2 weeks after ligature with progressive recovery beginning between W3 and W4. However, the largest fibres had not totally recovered by W15, contrasting with the disappearance of abnormal nociceptive reactions between W8 and W10. Although the damage to unmyelinated fibres is of importance, the abnormal pain-related behaviours seen in these rats appeared to be closely linked to the presence of both degenerative and regenerative changes in the A delta-range fibres, which did not necessarily correspond to initial A delta fibres.

Alterations in the properties of hippocampal pyramidal neurons in the aged rat.

The electrophysiological and pharmacological properties of CA1 hippocampal pyramidal neurons were studied in slices from young (three to four months) and aged (25-32 months) Sprague-Dawley rats having previously performed two behavioral tasks. About 20% of the aged rats were impaired in either the spontaneous alternation task or the water maze task. Electrophysiological parameters were measured and compared in young and aged animals using intracellular recordings. No age-related differences were observed in membrane potential, input resistance, amplitude of action potentials or amplitude of calcium spikes. The amplitude and duration of individual afterhyperpolarizations following a single spike were unchanged. In contrast, the neuronal excitability was significantly decreased and the spike duration significantly enhanced in aged rats as compared to young rats. The comparison of afterhyperpolarizations (which follow a burst of spikes) between young and aged rats was more complex. An increase in the amplitude and duration of afterhyperpolarizations generally occurred in aged animals. However, this increase was not consistent among animals and was dependent on the holding potential of the neuron and on the number of action potentials used to trigger the afterhyperpolarization. The depolarizing effect of bath-applied carbachol, as well as the associated increase in membrane resistance were reduced in neurons from aged rats. In contrast, the effects of carbachol on the depression of synaptic events and the blockade of the afterhyperpolarizations were similar in young and aged animals. In addition, the amplitude of the slow cholinergic excitatory postsynaptic potential induced by stimulation of cholinergic afferents in the presence of physostigmine was also decreased in aged rats. Excitatory postsynaptic potentials and inhibitory postsynaptic potentials following electrical stimulation of stratum radiatum were compared. The amplitude and duration of excitatory postsynaptic potentials were increased in aged rats. The amplitude and duration of the fast inhibitory postsynaptic potential were not significantly affected in aged animals. In contrast, the duration of the slow inhibitory postsynaptic potential was decreased in aged rats. Since the mean baclofen-induced hyperpolarization was only slightly reduced in aged rats, the most likely explanation is a decrease in the release of GABA rather than an alteration in the postsynaptic response mediated by GABAB receptors. A statistically significant correlation was found between the degree of impairment in the spontaneous alternation task and the amplitude of the carbachol-induced depolarization.

Protein SP40,40-like Immunoreactivity in the Rat Brain: Progressive Increase With Age.

The pattern of distribution of SP40,40-like immunoreactive structures has been studied in the rat brain using a well-characterized polyclonal antibody raised against the SP40,40 protein. Protein SP40,40 is the human counterpart of the rat sulphated glycoprotein 2, whose mRNA shows widespread expression in the developing and mature brain. In young adult rats few immunoreactive structures were observed. Some immunoreactive neurons were found in the cingulate cortex, the arcuate and perifornical hypothalamic nuclei, as well as glial labelling in the hypothalamus. A striking increase in the number of immunoreactive cells was observed as a function of age. In 20 - 22-month-old rats, numerous immunoreactive cells were observed in the cingulate cortex, several thalamic and hypothalamic nuclei, the red nucleus, olivary nuclei, superior colliculus, and many cranial nerve nuclei. Whereas the immunoreactivity was restricted to a diffuse labelling of the cell bodies and processes in young rats, other forms of labelling were observed in aged rats: punctate cytoplasmic labelling and intensely stained granules with no visible cell membrane. A further increase in the density of the immunoreactive material was observed in 30 - 31-month-old rats. Double labelling experiments demonstrated that the SP40,40 immunoreactivity was almost exclusively located in neurons and not in glial cells (with the exception noted above). The distribution of SP40,40 immunoreactivity in aged rats did not coincide with the distribution of the microtubule-associated tau protein, OX42 or lipofuscin.

The spectrum of fiber loss in a model of neuropathic pain in the rat: an electron microscopic study.

Recently, Bennett and Xie reported that when the sciatic nerve of the rat is ligated loosely, the rat develops a pain syndrome with many features similar to those observed in neuropathic pain states in man. Anatomical and physiological studies to date indicate that the major pathology is a loss of large diameter myelinated fibers distal to the ligatures, with more subtle changes in small myelinated fibers. With a view to evaluating possible changes in the unmyelinated fibers, we have performed an electron microscopic analysis of the sciatic nerve 2 weeks after four ligatures were applied, at which time the animals displayed profound hyperalgesia and mechanical and thermal allodynia. Cross-sectional photomontages of regions proximal and distal to the ligatures were studied. Consistent with light microscopic and electrophysiological studies, we found a near complete loss of large myelinated fibers distal to the ligatures. Phagocytosis of large fibers was common. There was also considerable variation in the damage to small myelinated fibers. In some fascicles many small (less than 3 microns) myelinated axons remained; in other fascicles none could be detected. Importantly, we also found significant changes in the unmyelinated fiber spectrum. Counts of unmyelinated axons revealed a 34% and 71% decrease in the distal compared to the proximal nerve, in the two rats studied. The large clusters of unmyelinated axons that characterize normal nerve (and the nerve proximal to the ligatures) were rarely found distally. Rather, many of the unmyelinated axons coursed singly or in very loose bundles. Many of the surviving axons were shrunken and distorted, although still in contact with Schwann cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative study of the effects of tianeptine and other antidepressants on the activity of medial septal neurons in rats anesthetized with urethane.

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.

Age-related changes in galanin-immunoreactive cells of the rat medial septal area.

Age-related changes in the cholinergic cells have been reported in the rat medial septal area. The neuropeptide galanin is colocalized with acetylcholine in the majority of the medial septal neurons. To assess possible age-related changes in the galanin-containing septal cells, we have examined, with immunohistochemical methods, the distribution pattern, density, and morphological features of galanin-containing cells in the rat medial septal nucleus (MS) and the nucleus of the diagonal band of Broca (DBB) in 1, 3-6, 9-12, 16-18, 24-27, and 28-30 month-old rats. A morphometric computerized analysis was also performed. In addition, the intensity of the immunolabelling was measured by densitometry. Galanin-like immunoreactivity (galanin-LI) was present in both the MS and the DBB. Our results clearly indicate a progressive age-related decrease in the number of galanin-positive cells throughout the MS-DBB complex. Our quantitative study revealed a significant loss of galanin-positive cells in the MS-DBB complex of 16-18 (50.4%), 24-27 (52.3%), and 28-30 (52.4%) month-old rats compared to 3-6 month-old animals. A non-significant reduction (28.6%) in galanin-LI cell number was observed in 3-6 month-old rats compared to 1 month-old animals. The morphometric analysis demonstrated a significant reduction (18%) in the surface of galanin-positive cells remaining in the 28-30 month-old group. Furthermore, a significant decrease in the immunolabelling intensity was consistently observed in animals of 16 month-old and older. To determine whether changes in galanin-positive cells were associated with cholinergic changes, the number of cells stained for acetylcholinesterase (AChE) was estimated in 3-6, 9-12, 16-18, and 24-27 month-old rats. There was a 43% decrease in the number of AChE-positive cells and a 71% loss of galanin-positive cells in 24-27 month-old rats compared to 3-6 month-old. The galanin-cell loss in the medial septal area was therefore associated with a parallel, although smaller, cholinergic septal cell loss.