Stereological estimates of the basal forebrain cell population in the rat, including neurons containing choline acetyltransferase, glutamic acid decarboxylase or phosphate-activated glutaminase and colocalizing vesicular glutamate transporters.

The basal forebrain (BF) plays an important role in modulating cortical activity and influencing attention, learning and memory. These activities are fulfilled importantly yet not entirely by cholinergic neurons. Noncholinergic neurons also contribute and comprise GABAergic neurons and other possibly glutamatergic neurons. The aim of the present study was to estimate the total number of cells in the BF of the rat and the proportions of that total represented by cholinergic, GABAergic and glutamatergic neurons. For this purpose, cells were counted using unbiased stereological methods within the medial septum, diagonal band, magnocellular preoptic nucleus, substantia innominata and globus pallidus in sections stained for Nissl substance and/or the neurotransmitter enzymes, choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) or phosphate-activated glutaminase (PAG). In Nissl-stained sections, the total number of neurons in the BF was estimated as approximately 355,000 and the numbers of ChAT-immuno-positive (+) as approximately 22,000, GAD+ approximately 119,000 and PAG+ approximately 316,000, corresponding to approximately 5%, approximately 35% and approximately 90% of the total. Thus, of the large population of BF neurons, only a small proportion has the capacity to synthesize acetylcholine (ACh), one third to synthesize GABA and the vast majority to synthesize glutamate (Glu). Moreover, through the presence of PAG, a proportion of ACh- and GABA-synthesizing neurons also has the capacity to synthesize Glu. In sections dual fluorescent immunostained for vesicular transporters, vesicular glutamate transporter (VGluT) 3 and not VGluT2 was present in the cell bodies of most PAG+ and ChAT+ and half the GAD+ cells. Given previous results showing that VGluT2 and not VGluT3 was present in BF axon terminals and not colocalized with VAChT or VGAT, we conclude that the BF cell population influences cortical and subcortical regions through neurons which release ACh, GABA or Glu from their terminals but which in part can also synthesize and release Glu from their soma or dendrites.

Cu, Zn- and Mn-superoxide dismutase levels in brains of patients with schizophrenic psychosis.

Impaired oxidative stress defense has been reported in blood of both drug-naïve and antipsychotic-treated patients suffering from schizophrenic psychosis, indicating the involvement of free radical metabolism in the pathogenetic processes of schizophrenia. In this study, the concentrations of two isoenzymes of superoxide dismutase (SOD), Cu, Zn- and MnSOD, were determined with ELISA in various cortical (frontal, parietal, temporal and occipital cortex) and subcortical areas (putamen, caudate nucleus, thalamus, and substantia innominata) of post-mortem brain tissue from patients diagnosed with a schizophrenia spectrum disorder and compared with those of controls. Post-mortem brain tissue from individuals without neuropsychiatric disorders served for control. Cu, Zn- and MnSOD levels were significantly increased in frontal cortex and substantia innominata of the index group, respectively. In all other areas both types of SOD remained virtually unchanged. Detection of SOD changes in the brain supports previous reports of alterations of antioxidant indices in blood cells of patients with schizophrenia and suggests a specific neuroanatomical distribution pattern of oxidative stress processes possibly related to the pathophysiology of schizophrenia.

Aggregates of cAMP-dependent kinase RIalpha characterize a type of cholinergic neurons in the rat brain.

Acetylcholine is synthesized by different types of neurons, showing a distinct biochemical phenotype. Aggregates of RIalpha regulatory subunit of cAMP-dependent protein kinases are visualized by immunohistochemistry only in some cholinergic neurons, since they tightly colocalize with two different markers, choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). These neurons are present mainly in brain areas related to the limbic system. None of the other regulatory subunits of cAMP dependent kinases colocalize with cholinergic markers.

Dementia with Lewy bodies: choline acetyltransferase parallels nucleus basalis pathology.

The biological substrate underlying the reduced cortical choline acetyltransferase (ChAT) in dementia with Lewy bodies (DLB) is incompletely understood. We compared cortical ChAT levels with Lewy body densities and neuronal loss in the nucleus basalis of Meynert (nbM) and cerebral cortex in six DLB, seven Alzheimer's disease (AD), and six control cases. We found greater neuronal loss in the nbM in DLB compared to AD (U = 9.500, p = 0.049). Mean ChAT levels in the cortex were lower in dementia patients than controls (t = 17.500, p = 0.001), and DLB cases had slightly lower ChAT levels than AD cases, but this difference was not significant (t = -0.332, p = 0.746). Overall, cortical ChAT levels correlated inversely with neuronal loss in the nbM (Spearman rank correlation coefficient = -0.53). The correlation between ChAT level and the combined factor of nbM LBs and neuronal loss was -0.59. A similar correlation between ChAT level and the combined factor of nbM neurofibrillary tangles and neuronal loss was -0.72. The correlation between ChAT and the combined factor of nbM LBs and neuronal loss was -0.81 when AD cases were excluded from the analysis. Local cortical pathology was not related to ChAT level. We conclude that neuronal loss and Lewy body formation in the nbM may contribute to the reduction in cortical ChAT in DLB.

Preservation of nucleus basalis neurons containing choline acetyltransferase and the vesicular acetylcholine transporter in the elderly with mild cognitive impairment and early Alzheimer's disease.

Immunocytochemistry for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) was used to examine the expression of these linked cholinergic markers in human basal forebrain, including cases with early stages of Alzheimer's disease (AD). Previous neurochemical studies have measured decreased ChAT activity in terminal fields, but little change or even increased levels of VAChT. To determine total cholinergic neuron numbers in the nucleus basalis of Meynert (nbM), stereologic methods were applied to tissue derived from three groups of individuals with varying levels of cognition: no cognitive impairment (NCI), mild cognitive impairment (MCI), and early-stage Alzheimer's disease (AD). Both markers were expressed robustly in nucleus basalis neurons and across all three groups. On average, there was no significant difference between the number of ChAT- (210,000) and VAChT- (174, 000) immunopositive neurons in the nbM per hemisphere in NCI cases for which the biological variation was calculated to be 17%. There was approximately a 15% nonsignificant reduction in the number of cholinergic neurons in the nbM in the AD cases with no decline in MCI cases. The number of ChAT- and VAChT-immunopositive neurons was shown to correlate significantly with the severity of dementia determined by scores on the Mini-Mental State Examination, but showed no relationship to apolipoprotein E allele status, age, gender, education, or postmortem interval when all clinical groups were combined or evaluated separately. These data suggest that cholinergic neurons, and the coexpression of ChAT and VAChT, are relatively preserved in early stages of AD.

Serotonergic input to cholinergic neurons in the substantia innominata and nucleus basalis magnocellularis in the rat.

The aim of the present study was to determine, at the light microscopic level, whether the serotonergic fibers originating from the dorsal raphe nucleus (B7), median raphe nucleus (B8) and ventral tegmentum (B9) make putative synaptic contacts with cholinergic neurons of the nucleus basalis magnocellularis and substantia innominata. For this purpose, we utilized: (i) the anterograde transport of Phaseolus vulgaris leucoagglutinin combined with choline acetyltransferase immunohistochemistry; (ii) choline acetyltransferase/tryptophan hydroxylase double immunohistochemistry; and (iii) the FluoroGold retrograde tracer technique combined with tryptophan hydroxylase immunohistochemistry. Following iontophoretic injections of Phaseolus vulgaris leucoagglutinin in the dorsal raphe nucleus, labeling was observed primarily in the ventral aspects of the nucleus basalis magnocellularis and in the intermediate region of the substantia innominata. When Phaseolus vulgaris leucoagglutinin was combined with choline acetyltransferase immunohistochemistry, a close association between the Phaseolus vulgaris leucoagglutinin-positive fibers and cholinergic neurons was observed, even though the majority of the Phaseolus vulgaris leucoagglutinin-immunoreactive terminals seemed to establish contact with non-cholinergic elements. Following Phaseolus vulgaris leucoagglutinin injection in the median raphe nucleus, very few labeled fibers with no evident close contact with nucleus basalis magnocellularis and substantia innominata cholinergic neurons were observed. After tryptophan hydroxylase/choline acetyltransferase double immunohistochemistry, a plexus of serotonergic (tryptophan hydroxylase-positive) fibers in the vicinity of choline acetyltransferase-immunoreactive neurons of the substantia innominata and nucleus basalis magnocellularis was observed, and some serotonergic terminals have been shown to come into very close contact with the cholinergic cells. Most of the tryptophan hydroxylase-immunoreactive terminals seem to establish contacts with non-cholinergic cells. Following FluoroGold injection in the nucleus basalis magnocellularis and substantia innominata, the majority of retrogradely labeled neurons was observed mainly in the ventromedial cell group of the dorsal raphe nucleus. In this area, a minority of the FluoroGold-positive neurons was tryptophan hydroxylase immunoreactive. These findings show that serotonergic terminals, identified in very close association with the cholinergic neurons in the substantia innominata and nucleus basalis magnocellularis, derive primarily from the B7 serotonergic cell group of the dorsal raphe nucleus, and provide the neuroanatomical evidence for a direct functional interaction between these two neurotransmitter systems in the basal forebrain.

NADPH-diaphorase histochemistry in the rat cerebral cortex and hippocampus: effect of electrolytic lesions of the nucleus basalis magnocellularis.

Unilateral or bilateral electrolytic lesions of the nucleus basalis magnocellularis (NBM) increased NADPH-diaphorase in the fronto-parietal cortex and in the CA1-CA3 fields of the hippocampus. NBM is the cholinergic basal forebrain nucleus supplying the fronto-parietal cortex but not the hippocampus. This increase was more remarkable at 4 weeks than at 2 weeks after lesioning. Monolateral or bilateral lesioning of the NBM increased to a similar extent NADPH-diaphorase. The number of neurons expressing NADPH-diaphorase was not statistically different between sham-operated and NBM-lesioned rats. These results indicate that similarly as reported in experimental damage of several brain areas, lesions of the NBM induce NADPH-diaphorase. The induction of this marker for nitric oxide synthase occurs both in the target of projections arising from the NBM such as the frontal cortex and in an area not directly supplied by NBM such as the hippocampus. Lesion-induced NADPH-diaphorase increase may contribute to neurodegenerative changes caused by damage of the NBM area.

Expression of tyrosine hydroxylase in magnocellular hypothalamic neurons of obese (fa / fa) and lean heterozygous (Fa / fa) Zucker rats.

In the magnocellular hypothalamic neurons (MCN) of normal rats, tyrosine hydroxylase (TH) is expressed in response to hyperosmotic stimulation and co-exists with vasopressin. The present study shows that both Zucker obese (fa / fa) and heterozygous lean (Fa / fa) rats express TH in MCN independently of an osmotic challenge. The lack of L-DOPA and aromatic-L-aminoacid decarboxylase in the MCN showed the absence of mechanisms necessary for catecholamine synthesis in these cells. Therefore, TH in MCN seems to be functionally inactive and is not involved in catecholamine abnormalities observed in these rats. All TH-immunoreactive MCN co-expressed vasopressin mRNA while a part of them co-expressed oxytocin mRNA. This suggests a mechanism of regulation of TH expression in MCN which is different in Zucker rats and in dehydrated normal rats.

Nitric oxide synthase in ventral forebrain grafts and in early ventral forebrain development.

Embryonic ventral forebrain (VFB) grafts to cortex contain neurons that synthesize acetylcholine and partially ameliorate behavioral deficits caused by excitotoxic damage to the nucleus basalis magnocelullaris in rats. An additional neurotransmitter, nitric oxide (NO), is synthesized by a subset of cholinergic neurons in rat ventral forebrain. If this neurotransmitter is expressed also by grafted cholinergic neurons (which include the embryonic medial septum and diagonal band), its functional contribution should be considered. Six to twelve months after transplantation of embryonic VFB tissue rats were sacrificed. Brain tissue was processed either for in situ hybridization of nNOS and neuropeptide Y (NPY) or for immunohistochemistry of choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS). Quantification of messenger ribonucleic acid (mRNA) for nNOS was performed with radioactively labeled probes (silver grains were counted) and a preliminary comparison was made of graft sections to sections of the ventral forebrain of developing rats. Plots of silver grain counts against cell size revealed similar patterns in the grafts and in the ventral forebrain of developing rats. The rates of expression of mRNA for nNOS in the grafts were intermediate between those of the ventral forebrain of postnatal day 19 and those of postnatal day 12. Double immunohistochemical labeling revealed that 45.87 + 8.26% of cells expressing ChAT also expressed nNOS in the grafts, significantly higher than 33.16 + 3.9% which was the rate of co-expression observed in the adult ventral forebrain. This study suggests that possible contribution of NO to graft-associated modulation of behavior should be examined.

Chronic ethanol ingestion produces cholinergic hypofunction in rat brain.

Alterations in cholinergic function due to prolonged ethanol exposure (up to 9 months) were assessed by choline acetyltransferase (ChAT) activity and high-affinity choline uptake (HAChU) in three brain regions of the Long-Evans rat: frontal cortex, parietal cortex, and region of the nucleus basalis of Meynert (NbM). No statistically significant changes were found in ChAT activity in the 3-month group; however, ChAT activity was decreased in both the frontal cortex (-32%) and NbM region (-22%) after 6 months of ethanol exposure. ChAT activity in the parietal cortex was increased 30% after 6 months. Nine months of exposure significantly decreased ChAT activity in all three brain regions. No significant differences were observed in high-affinity choline uptake after 3 months of ethanol exposure. However, after 6 months of ethanol exposure HAChU was decreased to 51% of control values in the frontal cortex. There was a simultaneous increase in HAChU to 43% and 178% of control values in the NbM and parietal cortex, respectively. However, choline uptake was significantly decreased in the frontal cortex and NbM region after 9 months of exposure. The results indicate a neurotoxic effect of prolonged intake of ethanol on the basal forebrain cholinergic projection system, which may cause impairment of cholinergic innervation of target areas of the basal nucleus complex.

Different distribution of dihydrolipoamide succinyltransferase, dihydrolipoamide acetyltransferase and ATP synthase beta-subunit in monkey brain.

The distribution of three mitochondrial enzymes: dihydrolipoamide succinyltransferase, dihydrolipoamide acetyltransferase, and beta-subunit of ATP synthase, were examined in the nucleus basalis of Meynert, substantia nigra, locus coeruleus, hippocampus, and cerebral cortex of monkey brain by immunocytochemical staining. Dihydrolipoamide succinyltransferase and dihydrolipoamide acetyltransferase had parallel distribution in the substantia nigra, but dihydrolipoamide acetyltransferase was rich in the locus coeruleus, nucleus basalis of Meynert, and especially in the hippocampus in comparison with dihydrolipoamide succinyltransferase. The ATP synthase beta-subunit was strikingly rich in many neurons of the locus coeruleus and cerebral cortex in comparison with dihydrolipoamide acetyltransferase and dihydrolipoamide succinyltransferase. These results show that these mitochondrial enzymes are not expressed synchronously in the neurons of brain, suggesting the differential regulation of mitochondrial enzymes and the heterogeneity of mitochondria.

An indirect cholinesterase inhibitor, metrifonate, increases neocortical EEG arousal in rats.

We investigated the ability of a cholinesterase inhibitor, metrifonate, to desynchronize cortical EEG activity. Metrifonate suppressed immobility-related high voltage spindling activity in young and aged rats at doses of 30 and 60 mg kg-1, p.o., and 10, 30 and 60 mg kg-1, p.o., respectively. The increase in EEG 1-20 Hz amplitude induced by scopolamine (0.2 mg kg-1, i.p.) was fully alleviated by metrifonate (30 and 100 mg kg-1, p.o.) and partially alleviated by a reference cholinesterase inhibitor, THA (3 and 6 mg kg-1, i.p.). Nucleus basalis (NB) lesions induced by quisqualic acid decreased frontal cortical choline acetyltransferase activity by 80% and increased cortical EEG slow waves. Metrifonate and THA did not reverse NB lesion-induced EEG abnormality. We conclude that metrifonate enhances cholinergic desynchronization of cortical EEG waves and that a severe defect of presynaptic NB cholinergic fibres limits the therapeutic effects of metrifonate.

Cyclooxygenase-2 induction in cerebral cortex: an intracellular response to synaptic excitation.

We have characterised the induction of the mitogen-inducible form of cyclooxygenase, COX-2, in the rat cerebral cortex in response to excitotoxin injection into the nucleus basalis. This model is associated with intense stimulation of the ascending pathway to the cerebral cortex, seizure activity, and subsequent ipsilateral cortical induction of various immediate early genes (IEGs), including c-fos, c-jun, and zif268, and ornithine decarboxylase enzyme activity and mRNA, all of which processes are sensitive to treatment with the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801. In this study we show that excitotoxin injection also causes a marked induction of COX-2 mRNA in ipsilateral cortex detectable at 1 h and peaking at 4 h, where COX-2 mRNA levels were 19 times those in unoperated animals. Levels of COX-2 mRNA remained significantly elevated at 24 h. The early induction of COX-2 at 1 h was also seen in sham-operated animals, but at 4 h the COX-2 mRNA level was significantly increased (4.4-fold) in animals injected with excitotoxin compared with sham-operated animals. The induction at this time point (4 h) was explored pharmacologically and found to be significantly attenuated by treatment with MK-801 (1.5 mg/kg), lamotrigine (10 mg/kg), which prevents presynaptic glutamate release by blocking voltage-sensitive Na+ channels, and the glucocorticoid dexamethasone (3 mg/kg), which has an indirect inhibitory effect on phospholipase A2 and COX activity.(ABSTRACT TRUNCATED AT 250 WORDS)

NGF-mediated synaptic sprouting in the cerebral cortex of lesioned primate brain.

In the present study, coronal brain sections of cortically devascularized non-human primates (Cercopithecus aethiops) were used to assess the lesion-associated synaptic loss, and the effect of exogenous nerve growth factor (NGF) in preventing or reversing this neurodegeneration. The sections were immunolabeled with antibodies against the synaptic marker protein synaptophysin (SYN), as well as choline acetyltransferase (ChAT) and parvalbumin (PV) markers that identify cholinergic neurons and interneurons, respectively. We found that, compared to sham-operated animals, in the lesioned vehicle treated animals SYN immunoreactivity near the lesioned site in the frontoparietal cortex was decreased by 31%. Similarly, corrected optical density values of immunostained sections specific for ChAT in the nucleus basalis of Meynert (ipsilateral to the lesion) decreased by 20% and PV-immunoreactive neurons near the lesion decreased by 47%. In contrast, NGF-treated lesioned animals showed levels of SYN, ChAT, and PV immunoreactivity similar to sham controls. These results are consistent with previous studies and support the view that NGF may not only prevent neurodegenerative changes after neocortical infarction by protecting vulnerable neurons, but also is capable of inducing sprouting and synaptogenesis.

Microanatomical and electrophysiological changes of the rat dentate gyrus caused by lesions of the nucleus basalis magnocellularis.

The effect of unilateral or bilateral lesions of the nucleus basalis magnocellularis (NBM) on the dentate gyrus of the hippocampus were assessed using microanatomical and electrophysiological techniques. NBM is the main cholinergic basal forebrain nucleus that supplies the fronto-parietal cortex. Lesions were induced using the neurotoxin ibotenic acid or a radio-frequency system and did not affect glutamic acid decarboxylase activity both in the frontal cortex and in the hippocampus. At 4 weeks after lesioning, a loss of choline acetyltransferase (ChAT) activity and of ChAT-immunoreactive fibres was observed in the frontal cortex but not in the hippocampus and no changes in the density of granule neurons of the dentate gyrus or in the hippocampal long-term potentiation (LTP) were noticeable. At 8 weeks after lesioning the loss of both ChAT activity and of ChAT-immunoreactive fibres persisted in the frontal cortex of NBM-lesioned rats. Moreover, at this time a significant decrease in the density of granule neurons in the dentate gyrus accompanied by a reduced probability of dentate LTP induction were observed in both ibotenic acid- and radio-frequency-lesioned rats. These findings have shown that although NBM does not send direct cholinergic projections to the hippocampus, lesions of this cholinergic nucleus are accompanied by delayed neurodegenerative changes involving the dentate gyrus. This suggests the occurrence of indirect connections between NBM and the hippocampus, the functional relevance of which should be explored.

Increased number of NADPH-d-positive neurons within the substantia innominata in Alzheimer's disease.

NADPH-diaphorase histochemistry labels neurons containing nitric oxide synthase, the synthesizing for nitric oxide within the central nervous system. Quantitation revealed a statistically significant increase in the density of intensely (type 1) and moderately (type 2) but not lightly (type 3) NADPH-diaphorase stained neurons within the substantia innominata in AD as compared with age-matched controls. Increased numbers of NADPH-diaphorase neurons suggest excess nitric oxide production which may be neurotoxic to surrounding cholinergic neurons within the substantia innominata in Alzheimer's disease.

In vivo measurement of acetylcholinesterase activity in the brain with a radioactive acetylcholine analog.

A novel method for visualization of brain acetylcholinesterase (AChE) in vivo has been developed. Following intravenous administration of a radiolabelled acetylcholine analog, N-methyl-3-piperidyl acetate, there was very good agreement between the distribution of radioactivity and AChE activity in the brain of rat and monkey. The method would be applicable for in vivo studies of human brain AChE activity in disorders of central cholinergic systems such as Alzheimer's disease.

An immunohistochemical study of cathepsin E in Alzheimer-type dementia brains.

We studied the immunohistochemical localization of cathepsin E (cath E) in the brains of patients with Alzheimer disease (AD) and control brains. In the normal brain cathepsin E immunoreactivity was detectable in a small number (below 5%) of neocortical and hippocampal neurons. In AD brains cathepsin E antigen was revealed in most large cortical and hippocampal pyramids and in neurons of the Nuc. basalis of Meynert. Cathepsin E was also present in cerebral microvessels, microglia, and in senile plaques. The enzyme might play roles in the process of neurodegeneration taking place in AD.

Restoration of cognitive abilities by cholinergic grafts in cortex of monkeys with lesions of the basal nucleus of Meynert.

Three groups of marmosets were trained to perform a series of visual discrimination tasks in a Wisconsin General Test Apparatus. Two groups then received bilateral lesions of the basal nucleus of Meynert using the excitotoxin N-methyl-D-aspartate and were found to be severely impaired on relearning a visual discrimination first learnt prior to surgery. One lesioned group then received grafts of acetylcholine-rich tissue dissected from the basal forebrain of fetal marmosets. Three months later the marmosets with lesion alone remained impaired on a number of retention and reversal tasks whereas the transplanted animals were no longer significantly impaired. Histological examination of the brains indicated that all lesioned animals had sustained substantial loss of the cholinergic neurons of the basal nucleus of Meynert (assessed by nerve growth factor receptor immunoreactivity) and that the lesion-alone animals showed marked loss of the cholinergic marker acetylcholinesterase in the dorsolateral frontal and parietal cortex. All transplanted animals had surviving graft tissue (visualized by Cresyl Violet staining, dense acetylcholinesterase staining and the presence of a limited number of nerve growth factor receptor-immunoreactive neurons) in the neocortex and 5/6 transplanted animals showed near complete restitution of acetylcholinesterase staining in frontal and parietal cortex. Examination of individual animal data showed that the animal without this restitution performed very poorly. The performance of the remaining transplanted animals was significantly better than that of the animals with lesion alone. There was a significant positive correlation between the degree of acetylcholinesterase staining and good performance on tasks sensitive to frontal lobe damage. These results demonstrate that acetylcholine-rich tissue transplanted into the neocortex of primates with damage to the cholinergic projections to the neocortex can produce substantial restitution of function provided that an appropriate level of interaction between graft and host tissue is achieved.

Neocortical infarction in subhuman primates leads to restricted morphological damage of the cholinergic neurons in the nucleus basalis of Meynert.

The aim of the present study was to investigate the long-term effect of cortical infarction on the subhuman primate (Cercopithecus aethiops) basal forebrain. The lesion, carried out by cauterizing the pial blood vessels supplying the left fronto-parieto-temporal neocortex, induced retrograde degenerative processes within the ipsilateral nucleus basalis of Meynert. The morphometrical analysis revealed that significant shrinkage of cholinergic neurons and loss of neuritic processes were localized within the intermediate regions of the nucleus basalis. The average cross-sectional areas of choline acetyltransferase-immunoreactive neurons in the intermedio-ventral (Ch4iv) and intermedio-dorsal (Ch4id) nucleus basalis were decreased to 62.5 +/- 9.5 and 58.0 +/- 8.6%, respectively, of the sham-operated values. Although an apparent loss of Nissl-stained magnocellular neurons in Ch4iv and Ch4id was found by applying a quantitative analysis based on a perikaryal-size criterion, data obtained by the quantification of immunostained material failed to reveal any significant decrease of cholinergic cell density. Results are discussed in view of future application of this ischemic model to study processes of retrograde degeneration following cortical target removal and to assess potential neurotrophic and neuroprotective properties of pharmacologic agents.