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Digoxigenin-labeled riboprobes and in situ hybridization of choline-O-acetyltransferase mRNA, both alone and in combination with immunohistochemical procedures for the synthetic enzyme of acetylcholine, were used to map the topography of putative cholinergic neurons in the rat central nervous system. Only the anti-sense riboprobe yielded specific labeling, which was absent in brain sections processed with sense riboprobe. Telencephalic neurons demonstrating the mRNA for choline-O-acetyltransferase and choline-O-acetyltransferase-like immunoreactivity were found in the caudate-putamen nucleus, nucleus accumbens, olfactory tubercule, Islands of Calleja complex, medial septal nucleus, vertical and horizontal limbs of the diagonal band, substantia innominata, nucleus basalis, and nucleus of the ansa lenticularis, as well as occasionally in the amygdala. Neurons in the cerebral cortex, hippocampus, and primary olfactory structures did not demonstrate hybridization signal, even though some cells in those areas were observed to exhibit choline-O-acetyltransferase-like immunopositivity. Thalamic cells were devoid of hybrido- and immunoreactivity, with the exception of several neurons located primarily in the ventral two-thirds of the medial habenula. A few cell bodies labeled with riboprobe and co-localizing choline-O-acetyltransferase-like immunopositivity were found in the lateral hypothalamus, caudal extension of the internal capsule, and zona incerta. Neurons in the pedunculopontine and laterodorsal tegmental nuclei evinced moderate hybridization signal, whereas cells of the parabigeminal nucleus were very weakly reactive. In contrast, motor neurons of the cranial nerve nuclei demonstrated high levels of choline-O-acetyltransferase mRNA and choline-O-acetyltransferase-like immunoreactivity. Putative cholinergic somata in the ventral horns and intermediolateral cell columns of the spinal cord and around the central canal were also labeled with riboprobe. It is concluded that hybridocytochemistry with digoxigenin-labeled riboprobes confirms the existence of cholinergic neurons in most of the neural regions believed to contain them on the basis of acetylcholinesterase pharmacohistochemistry and choline-O-acetyltransferase immunocytochemistry, with the prominent exceptions of the cerebral cortex, hippocampus, olfactory bulb, anterior olfactory nucleus, and caudal raphe nuclei, which apparently do not possess neurons expressing detectable levels of the mRNA for the synthetic enzyme of acetylcholine.

Digoxigenin-labeled RNA probes and in situ hybridization histochemistry were used to examine choline acetyltransferase gene expression in the rat central nervous system. Hybridization signal was present only in brain sections processed with the antisense riboprobe. The sense probe did not yield labeling, further validating the specificity of tissue reactivity. Telencephalic neurons containing the mRNA for the cholinergic synthetic enzyme were found in the caudate-putamen nucleus, nucleus accumbens, olfactory tubercule, islands of Calleja complex, medial septal nucleus, vertical and horizontal limbs of the diagonal band, substantia innominata, nucleus basalis, and nucleus of the ansa lenticularis. Some somata evincing hybridization signal were observed in the anterior amygdalar area, and an occasional such cell was seen in the basolateral and central amygdalar nuclei. Neurons in the cerebral cortex, hippocampus, and primary olfactory structures did not demonstrate hybridocytochemically detectable amounts of choline acetyltransferase mRNA. Thalamic cells were devoid of reactivity, with the exception of several neurons located primarily in the ventral two-thirds of the medial habenula. A few somata labeled with riboprobe were found in the lateral hypothalamus, caudal extension of the internal capsule, and zona incerta. Neurons in the pedunculopontine and laterodorsal tegmental nuclei were moderately reactive, whereas cells of the parabigeminal nucleus exhibited a very weak hybridization signal. No somata in the brainstem raphe nuclei, including raphe obscurus and raphe magnus, were observed to bind riboprobe. In contrast, motor neurons of the cranial nerve nuclei demonstrated relatively large amounts of choline acetyltransferase mRNA. Putative cholinergic somata in the ventral horns and intermediolateral cell columns of the spinal cord were also labeled with riboprobe, as were a few cells around the central canal. We conclude that hybridocytochemistry with digoxigenin-labeled riboprobes confirms the existence of cholinergic neurons (i.e. those that synthesize and use acetylcholine as a neurotransmitter) in most of the neural regions deduced to contain them on the basis of previous histochemical and immunocytochemical data. Notable exceptions are the cerebral cortex and hippocampus, which do not possess neurons expressing detectable levels of choline acetyltransferase mRNA.

Hyperthyroidism, induced in rat pups by the daily intraperitoneal administration of 1 microgram/g body weight triiodothyronine, facilitated the development of ChAT fiber plexuses in brain regions innervated by basal forebrain cholinergic neurons, leading to an earlier and increased expression of cholinergic markers in those fibers in the cortex, hippocampus and amygdala. A similar enhancement was seen in the caudate-putamen complex. This histochemical profile was correlated with an accelerated appearance of ChAT-positive telencephalic puncta, as well as with a larger total number of cholinergic terminals expressed, which persisted throughout the eight postnatal week, the longest time examined in the present study. Hypothyroidism was produced in rat pups by adding 0.5% propylthiouracil to the dams' diet beginning the day after birth. This dietary manipulation resulted in the diminished expression of ChAT in forebrain fibers and terminals. Hypothyroid treatment also reduced the quantity of ChAT puncta present during postnatal weeks 2 and 3, and, from week 4 and continuing through week 6, the number of ChAT-positive terminals in the telencephalic regions examined was actually less than the amount extant during the former developmental epoch. Immunostaining for nerve growth factor receptor (NGF-R), which is associated almost exclusively with ChAT-positive somata and fibers in the basal forebrain, demonstrated a different time course of postnatal development. Forebrain fibers and terminals demonstrating NGF-R were maximally visualized 1 week postnatally, a time at which these same neuronal elements evinced minimal ChAT-like immunopositivity. Thereafter and correlated with increased immunoreactivity for ChAT, fine details of NGF-R stained fibers were observed less frequently. Although propylthiouracil administration decreased NGF-R immunodensity, no alteration in the development of that receptor was observed as a function of triiodothyronine treatment. Cholinergic terminals in the ventrobasal thalamus, which derive from ChAT-positive neurons in the pedunculopontine and laterodorsal tegmental nucleus, were unaffected by either hyperthyroid or hypothyroid conditions. These cells also do not demonstrate NGF-R. We conclude from these experiments (1) that cholinergic fiber plexuses eventually exhibiting ChAT positivity in the telencephalon demonstrate NGF-R prior to the cholinergic synthetic enzyme, (2) that susceptibility to thyroid hormone manipulations may involve sensitivity to NGF, at least in some forebrain cholinergic systems and (3) that the effects of thyroid hormone imbalances on brain cholinergic neurons are regionally selective.

The compound 2,5-hexanedione (HD) produces axonopathies in peripheral nerves characterized by selective accumulation of neurofilaments. Its direct actions on neurotransmitter-specific neurons in the brain are unknown. In an attempt to address this latter issue, we infused HD into the fimbria and evaluated histochemically and immunohistochemically possible structural alterations in cholinergic neurons projecting from the basal nuclear complex to the hippocampus. Putative cholinergic fibers expressing nerve growth factor receptor and acetylcholinesterase showed increases in caliber and perturbations in trajectories 2-4 days following HD treatment. Similar morphologic changes were observed in neuronal elements processed for the 68 kDa neurofilament protein. At 7 days, short collateral ramifications appeared in many cholinergic axons that were suggestive of neurite outgrowth. Correlated with these fiber alterations was a transient reduction in the number of medial septal and diagonal band somata expressing choline acetyltransferase, which returned to control levels within 6 weeks following HD treatment. These data support the view that neurofilaments play an important, perhaps cytoarchitecturally stabilizing, role in regulating axonal morphology in certain populations of cholinergic neurons.

Both acetylcholinergic (ACh) and dopaminergic (DA) systems have been found to be crucial for the maintenance of accurate cognitive performance. In a series of studies examining those aspects of cognitive function revealed by the radial-arm maze, we have found that these two neurotransmitter systems interact in a complex fashion. Choice accuracy deficits in the radial-arm maze can be induced by blockade of either muscarinic- or nicotinic-ACh receptors. The choice accuracy deficit induced by blockade of muscarinic receptors with scopolamine can be reversed by the DA receptor blocker, haloperidol. The specific DA D1 blocker SCH 23390 also has this effect, whereas the specific D2 blocker raclopride does not, implying that it is D1 blockade that is critical for reversing the scopolamine effect. On the other hand, the choice accuracy deficit induced by nicotinic blockade with mecamylamine is potentiated by haloperidol. This effect is also seen with the D2 antagonist raclopride, but not with the D1 antagonist SCH 23390, implying that it is the D2 receptor which is important for the potentiation of the mecamylamine effect. The relevance of the D2 receptor for nicotinic actions on cognitive function is emphasized by the finding that the selective D2 agonist LY 171555 reverses the choice accuracy deficit caused by mecamylamine. Nicotinic and muscarinic blockade are synergistic in the deficit they produce. Antagonist doses subthreshold when given alone produce a pronounced impairment when given together. This latter deficit can be reversed by the D2 agonist LY 171555. These studies have outlined the complex nature of ACh-DA interactions with regard to cognitive function. Possible neural circuits for these interactions are discussed. The effectiveness of these selective DA treatments in reversing cognitive deficits due to ACh underactivation suggests a novel approach to treating cognitive dysfunction in syndromes such as Alzheimer's disease.

Choice accuracy performance in the radial-arm maze is dependent upon the integrity of both the nicotinic and muscarinic cholinergic receptors. Pharmacological blockade of either of these subtypes of cholinergic receptors with mecamylamine or scopolamine impairs choice accuracy in the radial-arm maze. We have previously demonstrated that the performance deficit caused by muscarinic blockade is exacerbated in at least an additive fashion by coadministration of the nicotinic antagonist, mecamylamine. In the present study, it was found that mecamylamine and scopolamine act together in a greater than additive fashion in disrupting radial-arm maze choice accuracy. When doses of these drugs which do not by themselves cause significant impairments in choice accuracy are given together, they induce a pronounced impairment. Previous results have shown that the adverse effects of nicotinic blockade could be reversed by the dopaminergic D2 agonist LY 171555. In this study, this drug was found to attenuate the cognitive impairment caused by combined nicotinic and muscarinic blockade. On the other hand, the dopaminergic D1 antagonist SCH 23390 which has previously been shown to reverse the adverse effects of muscarinic blockade was not found in this study to attenuate the impairment of combined nicotinic and muscarinic blockade. Since combined nicotinic and muscarinic blockade approximates generalized cholinergic underactivation, treatments like LY 171555, which attenuate the adverse effects of this combined blockade, may be useful in treating syndromes like Alzheimer's disease, which are characterized by generalized cholinergic loss.

The origins and acetylcholinesterase (AChE, EC 3.1.1.7) content of neurons projecting to the AChE-rich basolateral amygdala were studied by infusing Evans Blue (EB), a retrogradely transported fluorescent label, into that neural region and, following microscopic evaluation of labelled somata, staining the same tissue sections for AChE according to the pharmacohistochemical regimen. The following basal forebrain areas contained cells labelled with EB: the lateral preoptic area, ventral pallidum, nuclei of the diagonal band, medial septal nucleus, bed nucleus of the stria terminalis, and substantia innominata. The majority of the basal forebrain neurons projecting to the basolateral amygdala stained intensely for AChE, suggesting that they were cholinergic. In the brainstem, EB-labelled neurons staining intensely for AChE were found less frequently, but a few were observed in the nucleus tegmenti pendunculopontis, locus ceruleus, subcerulear region, and reticular formation. Cells accumulating EB after basolateral amygdala infusion but demonstrating no, weak, or moderate AChE activity were seen in the orbitofrontal, anterior cingulate, temporal, and insular cortices; the mediodorsal, paraventricular, and parataenial nuclei of the thalamus; the periventricular gray substance; the ventromedial mesencephalic tegmentum; the lateral and compact portions of the substantia nigra; the dorsal raphe; the dorsal tegmental nucleus; and the dorsal parabrachial nucleus. On the basis of staining intensity, intracellular organization of the AChE reaction product, and previous results in the literature, we conclude that the major cholinergic input to the basolateral amygdala derives from the basal forebrain.

In order to characterize further the origin of striatal afferents, adult rats underwent a unilateral intrastriatal infusion of 30% Evans Blue solution (0.2 - 0.5 mul). Labeled neurons were observed in ipsilateral substantia nigra, thalamus, cortex, ventromedial mesencephalic tegmentum and dorsal raphe. Several labeled neuronal somata, albeit considerably fewer than seen ipsilaterally, were found also in the contralateral substantia nigra and ventral tegmental area, a result contrary to previously reported findings in the rat. Control injections restricted to cortex overlying striatal target site did not result in similar labeling, and corpus callosum transection prior to intrastriatal injection did not prevent labeling of contralateral substantia nigra. These findings indicate that rats, like cats, have a sparse crossed nigrostriatal pathway.

The histopathologic effects of different doses of ethylcholine mustard aziridinium ion infused into the caudate-putamen complex or nucleus basalis were evaluated in rats. Although no non-specific tissue damage was observed at the lowest doses of ethylcholine mustard aziridinium ion examined--0.01 nmol in 1-microliter vehicle and 0.02 nmol in 2-, 5-, and 10-microliters vehicle in both the striatum and nucleus basalis--minimal but definite non-selective pathology, characterized by gliosis and loss of all neuronal elements in the region affected by the nitrogen mustard, was observed in both targets at a dose of 0.02 nmol 1 microliter and more severely at all doses containing 0.05 and 0.1 nmol ethylcholine mustard aziridinium ion. At doses of ethylcholine mustard aziridinium ion containing 0.2 nmol of the cytotoxin and greater amounts, non-specific cell loss in intact tissue and extensive cavitation became increasingly the most prominent histologic features of drug action. No statistically significant effects of ethylcholine mustard aziridinium ion on striatal choline acetyltransferase activities were found until doses of 0.4 nmol/1 microliter or greater were injected, concentrations of the cytotoxin at which appreciable non-specific pathology was also observed. Levels of dopamine in the caudate-putamen nucleus were reduced by comparatively greater amounts than choline acetyltransferase at doses of 2.5 nmol/2 microliters, 5.0 nmol/2 microliters and 10 nmol/2 microliters cytotoxin, but a significant effect of ethylcholine mustard aziridinium ion on striatal L-glutamate decarboxylase activity was found only at a dose of 10 nmol/2 microliters. As no dose of ethylcholine mustard aziridinium ion was found that reduced choline acetyltransferase without producing considerable non-specific tissue destruction, the usefulness of the cytotoxin in studying the behavioral and physiological consequences of selective cholinergic hypofunction in the brain must be questioned.

The effects of xylamine (N-2-chloroethyl-N-ethyl-2-methylbenzylamine) on catecholamine content and uptake in rat brain were examined after systemic administration. Four hours after doses of 10 to 50 mg/kg i.p. of xylamine, norepinephrine uptake in synaptosomal preparations from cortex was reduced, but dopamine uptake in striatal preparations was unaffected. The levels of norepinephrine in the cortex and hypothalamus were depressed over this dose range, whereas dopamine levels in the striatum again were unaltered. The depletion of norepinephrine from the cortex and hypothalamus was also seen histologically by fluorescence histochemistry. When brain tissue was examined 10 days after a single 25 mg/kg dose, the depletion persisted in cortex and hypothalamus, and although there was a decrease in fluorescence intensity there was no clear evidence of a decreased number of catecholamine-containing processes that would have indicated neurotoxic effects. There was also no gliosis (an indicator of neuronal degeneration) in any of the brain areas or damage to the blood brain barrier. Thus, in contrast to 6-hydroxydopamine, xylamine appears to have selective action on norepinephrine systems and does not appear to be a nonselective neurotoxin.

In the zebra finch (Poephila guttata), horseradish peroxidase injected into or near Area X of the lobus parolfactorius (LPO) is transported to cell bodies in ipsilateral hyperstriatum ventrale pars caudale (HVc), area ventralis of Tsai (AVT), and nucleus tegmenti pedunculo-pontinus, pars compacta (TPc). Area X, LPO, and paleostriatum augmentatum (PA) all contain a dense network of catacholamine-containing axons and nerve terminals, as determined in histofluorescence studies. Cell bodies in AVT and TPc contain catacholamines; lesions of TPc greatly reduce or abolish catacholamine fluorescence in PA, and a lesion of AVT eliminates histofluorescence in LPO, icluding Area X. The anatomical location and catecholaminergic projection from AVT suggest that LPO-Area X may be the avian homolog of the mammalian nucleus accumbens, olfactory tubercle, and/or rostromedial caudate.

Various doses of the neurotoxin 6-hydroxydopamine (6-OHDA) were infused into the substantia nigra-ventromedial tegmental area, caudateputamen nucleus, and red nucleus. A maximum of four successively occurring zones of neuropathology could by detected at all injection sites: (1) A zone of complete absence of neural, glial, and vascular elements due to tissue displacement by the cannula, (2) an area of glial cells, developing over time, which surrounded the cannula tract, (3) a region exhibiting virtually complete loss of neuronal elements, and (4) a zone of selective neuropathological reaction or cellular loss in which some neurons were affected and others were not. All investigators who have histochemically and/or histologically evaluated the effects of intracerebrally administered 6-OHDA agree on the existence and genesis, some non-selective process, of the first three zones. Although some scientists maintain that the fourth zone is where 6-OHDA operates selectively, data are presented in this report that other, well-established processes can account for neuropathology beyond zone 3. Prominent among these are retrograde and anterograde degeneration, ischemia as a function of interruption of blood supply, and nonselective traumatization. In addition, the topography of the injected site (e.g., morphology of affected neurons, degree of myelination) was found to be as important a determinant of the locus of tissue damage as the presumed selectivity of 6-OHDA's action. Furthermore, monoamine oxidase inhibition by nialamide did not appear to increase the neurotoxicity of 6-OHDA. Similarly, 1-(2,5-dihydroxy-4-methylphenyl)-2-aminopropane was not a more potent cytotoxin than 6-OHDA even though this new neurotoxin has a propane side chain which renders it immune to monoamine oxidase. These observations, taken together, suggest that considerable cautioon should be exercised in interpreting data from experiments in which 6-OHDA, or related neurotoxins, are used to uncover catecholaminergic mechanisms of behavior and other functional processes.

The activity of reduced nicotinamide adenine dinucleotide (NADH)-diaphorase was examined histochemically in the amygdala, cortex and sublenticular substantia innominata (nucleus basalis of Meynert) of patients with Alzheimer's disease and senile dementia of the Alzheimer type (SDAT). Senile plaques were characterized by increased enzyme levels and the presence of astrocytes highly reactive for NADH-diaphorase. In the sublenticular substantia innominata, the number of neurons positive for NADH-diaphorase was reduced in both Alzheimer's disease and SDAT, a result paralleled by a reduction of Nissl-stained cells, and this pathology was accompanied by an increase in the number of astrocytes. Intact substantia innominata somata in the former dementia, however, showed essentially normal levels of the enzyme, whereas in the SDAT patients, an abnormal distribution of NADH-diaphorase was observed frequently. It is proposed that the increased NADH-diaphorase associated with senile plaques and their accompanying astrocytes may be linked, in part, to the increased astrogliosis and decrease of neurons in the basal forebrain and that neuropathologic differences may exist between Alzheimer's disease and SDAT in terms of energy metabolism.

The cholinergic innervation of the interpeduncular nucleus was investigated by use of fluorescent tracer histology in combination with choline-O-acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) pharmacohistochemistry. Following propidium iodide or Evans Blue infusion into the interpeduncular nucleus, brains were processed for co-localization of transported fluorescent label and ChAT and AChE. Control infusions of tracers were made into the ventral tegmental area. In order to delimit the course of putative cholinergic afferents to the interpeduncular nucleus from extra-habenular sources, knife cuts surrounding the habenular nuclei were performed. Somata containing propidium iodide that were highly immunoreactive for ChAT were found primarily in the vertical and horizontal limbs of the diagonal band, the magnocellular preoptic area, and the dorsolateral tegmental nucleus, also referred to as the laterodorsal tegmental nucleus. A few such co-labeled somata were also detected in the medial septal nucleus, substantia innominata, nucleus basalis, and pedunculopontine tegmental nucleus. A good correlation was observed between intensely-staining, AChE-containing and ChAT-positive neurons projecting to the interpeduncular nucleus from the aforementioned structures. Although the medial habenula contained numerous cells demonstrating transported label following interpeduncular infusion of fluorescent tracers, the ChAT-positivity associated with somata in that nucleus was weak compared to ChAT-like immunoreactivity in known cholinergic neurons in the basal forebrain and brainstem. Knife cuts that separated the habenular nuclei from the stria medullaris and neural regions lateral and posterior to those nuclei while leaving the fasciculus retroflexus intact resulted in a reduction of ChAT-like immunoreactivity in the medial habenular nucleus, fasciculus retroflexus, and interpeduncular nucleus. These data suggest (1) that the cholinergic innervation of the interpeduncular nucleus derives primarily from ChAT-positive cells in the basal forebrain and dorsolateral tegmental nucleus and (2) that putative cholinergic fibers having their origin in the medial habenula, if they exist, constitute a minor portion of the cholinergic input to the interpeduncular nucleus.

The morphologies, intercellular organization, and cortical projection patterns of putative cholinergic neurons in the basal forebrain of the rat were examined by use of fluorescent tracer histology in combination with the pharmacohistochemical regimen for acetylcholinesterase (AChE). Intensity staining AChE-containing cells projecting to frontal sensorimotor (Area 10), parietal (Area 2), and temporal (Area 4) cortices were found ipsilaterally in nucleus preopticus magnocellularis, in nucleus basalis, and in association with the substantia innominata, the ansa lenticularis, and the lateral hypothalamic area; an essentially rostrocaudal topography was observed for these projections. AChE-containing pathways to cingulate (Area 29) and visual (Area 17) cortices derived from ipsilateral somata associated with the vertical and horizontal limbs of the diagonal band, nucleus preopticus magnocellularis, rostral portions of nucleus basalis, and the substantia innominata. Neurons innervating Area 29 were generally located more rostrally than those giving rise to AChE afferents to Area 17. The vast majority of cells appeared to innervate relatively discrete areas of the cortex. Evidence for collateralization was found only in neurons projecting to visual and cingulate cortices, and these represented only 3.2% of the cells providing AChE afferents to Areas 17 and 29. The basal forebrain AChE projection cells were typically large (greater than 25 micron in maximum cell body extent), and their somata were predominantly oval, with lesser proportions being fusiform or triangular. Many were organized in clusters, particularly in nucleus basalis.

The distribution of acetylcholinesterase (AChE, EC3.1.1.7) within extrapyramidal and related structures was studied in 4 monkeys following the i.m. administration of bis-(1-methylethyl) phosphorofluoridate (di-isopropylfluorophosphate: DFP). In 1 animal, sacrificed 4 hr after the injection of 0.43 mg/kg C, AChE is virtually absent in all structures. In the other 3 animals, which received 0.20 mg/kg DFP 10, 12 and 18 hr prior to sacrifice, AChE activity is greatly reduced in the neuropil of those structures which normally show intense AChE activity in pharmacologically unmanipulated monkeys. As a consequence of the lower background AChE activity in the latter 3 DFP-treated monkeys, the perikarya and processes of several groups of neurons can be readily identified. The perikarya and processes of two types of neurons of the neostriatum, representing a small percentage of all neurons in this structure, are intensely stained. They apparently correspond to the chromatic giant aspiny neurons and the achromatic medium-size "spidery aspiny" neurons. Most perikarya and processes of the neurons of the medial and lateral divisions of the pallidum, as well as the morphologically similar neurons of the pars reticulata of the substantia nigra, display light to moderate AChE activity. The pars compacta of the substantia nigra contains a small number of intensely stained elongated and triangular neurons and numerous moderately stained ovoid neurons whose processes are very lightly reactive. The cell bodies of the neurons of the subthalamic nucleus and associated groups of neurons (nucleus ansae peduncularis) are moderately stained whereas the perikarya and processes of the subnucleus compactus of the pedunculopontine tegmental nucleus are intensely and moderately stained, respectively. Numerous large and multipolar neurons associated with the lenticular nucleus (intralamellar groups) or the pallidum (peripallidal groups) including the nucleus ansae lenticularis have somata and processes which show an intense AChE activity. Certain of the latter groups of neurons, partially interspersed with the neurons of the substantia innominata, correspond in part to the nucleus basalis of Meynert. The present data are compatible with the idea that the large AChE neurons of the neostriatum may be the source of the acetylcholinesterasic striatopallidal and strionigral fibers. The similarity between the neurons of the pallidum and those of the pars reticulata of the substantia nigra suggests that the latter structure may represent a caudal extension of pallidal tissue. Therefore, the nigrothalamic projection, which has been claimed to originate in the pars reticulata of the substantia nigra, would correspond to pallidothalamic projections.

A pharmaco-histochemical regimen was used to examine the morphology and internal organization of acetylcholinesterase (AChE, EC 3.1.1.7) neurons in brain areas--the caudate-putamen nucleus, nucleus accumbens, olfactory tubercule, and subtantia nigra--monoaminergically characterized in terms of their dopamine content. Intense, homogenous staining is produced in these neural regions by other histochemical protocols for AChE; individual AChE-containing neurons cannot be observed reliably or consistently. With the present technique, based on the differential regeneration of AChE in the separate subcellular compartments of the neuron (i.e., axon, dendrite, soma) after intramuscular injection of bis-(1-methylethyl)-phosphorofluoridate (di-isopropylfluorophosphate: DFP), it was shown that AChE was associated with neurons whose cell bodies lay within the brain areas studied. Although the significance of dopaminergic-cholinergic relationships in the caudate-putamen complex, nucleus accumbens, and olfactory tubercule could not be established on the basis of these new histochemical data, arguments were presented indicating that dopamine neurons in the zona compacta of the substantia nigra also contained AChE.

Acetylcholine (ACh) systems have been found to be crucial for the maintenance of accurate cognitive performance. A great variety of studies have shown that the muscarinic ACh receptor blocker scopolamine impairs choice accuracy in the radial-arm maze. Recently, it has been found that the nicotinic ACh receptor blocker mecamylamine also impairs radial-arm maze choice accuracy. In the present study, we investigated the effects of combined administration of these two ACh blockers. Scopolamine (0.15 mg/kg) and mecamylamine (10 mg/kg) each moderately impaired choice accuracy. Combined treatment with scopolamine and mecamylamine significantly decreased choice accuracy relative to either drug alone. This combination treatment lowered choice accuracy to chance levels. These data show that nicotinic and muscarinic blockade have at least additive effects in producing an anterograde memory deficit. Concurrent blockade of these two components of ACh systems may provide a better animal model of cognitive impairments due to the loss of cholinergic neurons, such as Alzheimer's disease.