c-ret regulates cholinergic properties in mouse sympathetic neurons: evidence from mutant mice

The search for signalling systems regulating development of noradrenergic and cholinergic sympathetic neurons is a classical problem of developmental neuroscience. While an essential role of bone morphogenetic proteins for induction of noradrenergic properties is firmly established, factors involved in the development of cholinergic traits in vivo are still enigmatic. Previous studies have shown that the c-ret receptor and cholinergic properties are coexpressed in chick sympathetic neurons. Using in situ hybridization we show now that a loss-of-function mutation of the c-ret receptor in mice dramatically reduces numbers of cells positive for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) in stellate ganglia of homozygous newborn animals. The number of neurons positive for tyrosine hydroxylase (TH) mRNA, the rate-limiting enzyme of noradrenaline synthesis, is reduced to a smaller degree and expression levels are not detectably altered. Already at embryonic day 16 (E16), ChAT and VAChT-positive cells are affected by the c-ret mutation. At E14, however, ChAT and VAChT mRNAs are detectable at low levels and no difference is observed between wildtype and mutant mice. Our data suggest that c-ret signalling is necessary for the maturation of cholinergic sympathetic neurons but dispensable for de novo induction of ChAT and VAChT expression.     

BDNF and CNTF regulate cholinergic properties of sympathetic neurons through independent mechanisms

Cultured neonatal sympathetic neurons can synthesize and corelease norepinephrine (NE) and acetylcholine (ACh). Evoked release of NE has an excitatory effect on the beat rate of cocultured cardiac myocytes while ACh release results in myocyte inhibition. Here we show that the cholinergic properties of the neurons and the relative level of NE and ACh corelease are modulated by neurotrophic factors. Brain-derived neurotrophic factor (BDNF) rapidly promoted ACh release in the absence of cholinergic differentiation activity and even in neurons that were predominantly noradrenergic. This increase in the cholinergic component of sympathetic cotransmission was sufficient for myocytes to display an overall inhibitory response to neuronal stimulation. In contrast, short-term growth in ciliary neurotrophic factor (CNTF) resulted in the upregulation of cholinergic and downregulation of noradrenergic markers without an effect on normal excitatory neurotransmission. Only once the cells had acquired a cholinergic phenotype did CNTF acutely promote the evoked release of the cholinergic vesicle pool. The results of this study indicate that BDNF and CNTF, acting through independent pathways, modulate NE and ACh cotransmission to regulate the level of sympathetic excitation or inhibition of cardiac myocytes.     

Developing sympathetic neurons express a neuronal trait before a catecholaminergic synthetic enzyme in vivo

How multiple mature phenotypic traits are regulated in developing neurons remains a central problem in developmental neurobiology. Mature sympathetic neurons express general neuronal epitopes, including neuron specific tubulin (NST) as well as markers involved in neurotransmitter synthesis including tyrosine hydroxylase (TH). To investigate the relationship between neuronal differentiation and neurotransmitter development, the order of appearance of NST and TH was determined in both chick and rat embryonic sympathetic ganglia by double-label immunohistochemistry. In differentiated ganglia, these two markers were expressed in virtually all sympathetic neurons examined. By contrast, at early stages of embryonic development in both chick and rat, sympathetic ganglia contained many NST-IR immunoreactive (IR) cells, but few precursors were TH-IR. With further development, more NST-IR cells were also TH-IR, and these two markers gradually became coexpressed with subsequent development. These data provide evidence that general neuronal and neurotransmitter differentiation events are separable and independently regulated during cellular diversification in the adrenergic lineage. © 1996 Wiley-Liss, Inc.     

Environmental co-regulation of substance P, somatostatin and neurotransmitter synthesizing enzymes in cultured sympathetic neurons

Mechanisms regulating peptide neurotransmitter metabolism were examined in dissociated cell cultures of the neonatal rat superior cervical ganglion (SCG). The pineal gland, a target of the SCG, produced a soluble factor (PCM) which increased substance P (SP) levels more than 15-fold in sympathetic neurons cultured in the presence of ganglion non-neuronal cells. Elimination of the non-neuronal cells decreased SP to negligible levels and abolished the stimulatory effects of PCM on SP expression. These observations suggest that ganglion non-neuronal cells stimulate sympathetic expression of SP, and that the pineal influences neuronal SP by acting on, or in concert with, ganglion support cells. PCM also influenced other neurotransmitter systems. In the presence of ganglion non-neuronal cells, PCM treatment increased cholineacetyltransferase (CHAC) and decreased tyrosine hydroxylase (TOH) and somatostatin (SO). By contrast, PCM treatment of pure neuronal cultures resulted in negligibleCHAC and SP levels and a doubling of SO with a small increase in TOH. In sympathetic neurons, SP expression may be associated with cholinergic development, whereas SO may be associated with noradrenergic phenotypic expression. Moreover, there is a reciprocal relationship between SP and SS expression by sympathetic neurons analogous toe previously described relationship between noradrenergic and cholinergic expression^17–19.     

CRF-like immunoreactivity selectively labels preganglionic sudomotor neurons in cat.

Immunohistochemical and neuronal tracing methods were used in cats to determine which type of postganglionic sympathetic neuron is innervated by preganglionic neurons which contain corticotrophin releasing factor-like immunoreactivity (CRF-LI). Preganglionic neurons with CRF-LI have their cell bodies at two restricted levels of the spinal cord and terminate in the stellate and lower lumbar ganglia. CRF-LI terminal baskets in stellate and lumbar ganglia surrounded cell bodies, 96-99% of which showed no tyrosine hydroxylase (TH)-LI (presumptive cholinergic neurons). Calcitonin gene-related peptide (CGRP)-LI was used to label the cholinergic ganglion cells which innervate sweat glands: 96-99% of those were confirmed as lacking TH-LI, while the remainder showed weak staining. Every one of over 6000 CRF-LI terminal baskets counted in 4 stellate and 6 lumbar ganglia was found to surround a cell body with CGRP-LI; conversely, 81-86% of the cell bodies showing CGRP-LI were surrounded by CRF-LI terminal baskets. In 3 cats, the retrograde tracer fluorogold was used to label postganglionic neurons projecting to the paw pads (a population which includes both cholinergic sudomotor neurons and noradrenergic vasoconstrictor neurons). Between 26 and 38% of the retrogradely labelled ganglion cells were surrounded by CRF-LI terminal baskets. We conclude that in cats, preganglionic sympathetic neurons with CRF-LI are sudomotor in function.     

Chick eye extract promotes expression of a cholinergic enzyme in sympathetic ganglia in culture

Previous studies have demonstrated that, in rat, individual sympathetic neurons can express both adrenergic and cholinergic biosynthetic enzymes in culture. Moreover, the levels of these enzymes can be regulated by factors present in their environment. In the present study, we sought to determine whether cultures of chick sympathetic neurons express both adrenergic and cholinergic enzymes, whether both enzymes are expressed in the same neurons, and whether the levels of these enzymes can be influenced by environmental factors. In our system, we tested one such factor found in embryonic eye extract (EEE) which has been shown to specifically increase the activity of the cholinergic enzyme choline acetyltransferase (ChAT) in cultures of chick parasympathetic neurons Varon et al., Brain Res., 173 (1979) 29-45; Nishi and Berg. J. Neurosci., 1 (1981) 505-513). At various times in vitro, cultures were analyzed using biochemical, immunocytochemical and autoradiographic techniques. We found that only those cultures of sympathetic neurons supplemented with EEE developed detectable levels of ChAT enzyme activity at 2 days, which increased significantly by 14 days in vitro. Supplementation with EEE did not affect the level of tyrosine hydroxylase (TH) activity. Furthermore, irrespective of nutrient medium, all neurons in all cultures contained TH immunoreactivity and possessed a high-affinity amine uptake system as demonstrated by autoradiography. These studies suggest that neurons of chick sympathetic ganglia can be influenced by factors present in EEE to express a cholinergic enzyme and that this enzyme is coexpressed by cells also exhibiting an adrenergic phenotype.     

High-affinity uptake of noradrenaline in quail dorsal root ganglion cells that express tyrosine hydroxylase immunoreactivity in vitro

During embryonic life, avian sensory ganglia contain cells with the potential to express, under appropriate experimental conditions, a number of properties characteristic of autonomic sympathetic neurons. Thus, cells capable of synthesizing noradrenaline (NA) from tyrosine differentiate when dorsal root ganglia (DRG) from 10-15 d embryonic quail are grown in culture (Xue et al., 1985a, b). In the present study, we show that cultures of DRG from 10 d embryos can take up 3H-NA by a high-affinity (Km = 1.0 microM), temperature-dependent process that can be inhibited by desmethylimipramine. By means of combined immunocytochemistry and autoradiography, it was demonstrated that the majority (70-80%) of the tyrosine hydroxylase (TH)-immunoreactive cells that developed in the cultures possessed a transport system for NA. Catecholamine (CA) uptake also occurred in a small, but relatively constant, number of TH-negative cells, but was absent from substance P-containing neurons. In contrast to TH, which appears only after 3-4 d in vitro, cells capable of taking up NA with high affinity were found in DRG cultures after only a few hours, and a small number (less than 0.5% of the total cell population) was detected in freshly removed, uncultured ganglia. Such cells did not react with antibodies directed against substance P or neurofilament proteins. We conclude that autonomic precursors are identifiable in a subset of non-neuronal DRG cells, prior to full expression of a noradrenergic phenotype, by their possession of a high-affinity uptake system for CA.     

Selective modulation of cholinergic properties in cultures of avian embryonic sympathetic ganglia

We have studied the expression of catecholaminergic and cholinergic phenotypes in sympathetic ganglia removed from 7- to 10-day-old quail embryos and grown in vitro under different conditions. Quantitative data were obtained by measuring the conversion of (³H) tyrosine and (³H) choline to catecholamines (CA) and acetylcholine (ACh), respectively. In explant cultures, large amounts of both neurotransmitters were synthesized from the onset, but CA generally predominated, the molar ratios of CA:ACh being, on average, of the order of 2:1. If the ganglia were dissociated before plating, there was a selective increase in ACh synthesis (three- to fivefold) such that the CA:ACh ratio fell strikingly. The early expression of the cholinergic phenotype appears to be species-specific in that, under identical conditions, dissociated cell cultures of newborn mouse superior cervical ganglia were overwhelmingly catecholaminergic (CA:ACh ratio of approximately 40:1) and ACh synthesis was only just detectable. Addition of veratridine (1.5 μM) either to explant or to dissociated cell cultures of embryonic quail sympathetic ganglia barely altered CA-synthesizing ability; in contrast, ACh synthesis and accumulation were stimulated about threefold. This effect, which we found to correspond to a quantitatively similar increase in the activity of choline acetyltransferase (ChAT), was completely blocked by tetrodotoxin, indicating that it was due to Na⁺-dependent depolarization. A preferential stimulation of ACh production was also observed when the concentration of K⁺ was raised to 20 mM. Veratridine treatment of cultures of presumptive sympathoblasts, in the form of sclerotome-associated neural crest cells, had identical effects. Our results reveal the quantitative importance of ACh-related properties in avian sympathetic ganglia from the earliest stages of their development and suggest that depolarization may be one of the factors selectively enhancing expression of the cholinergic phenotype during ontogeny. In these respects, the neurochemical differentiation of sympathetic neurons unfolds according to dissimilar scenarios in birds and mammals. © 1993 Wiley-Liss, Inc.     

In vivo role for CREB signaling in the noradrenergic differentiation of sympathetic neurons

Signaling pathways involving cAMP and CREB have been implicated in several aspects of sympathetic neuron differentiation. Here, we used in vivo loss-of-function approaches in both mouse and chick embryos to characterize the physiological role of cAMP/CREB. Whereas sympathetic neuron development proceeds normally in CREB-deficient mouse embryos, a decrease in noradrenergic differentiation (TH, DBH) was observed in chick sympathetic ganglia in response to ACREB, a dominant-negative CREB variant which interferes with the function of all CREB family members. In contrast, expression of the generic neuronal marker SCG10 was not affected by ACREB. As the decrease in noradrenergic gene expression is compensated at later stages of development and TH expression in differentiated neurons is not CREB-dependent, a transient role for CREB is proposed, accelerating noradrenergic but not generic neuronal differentiation of sympathetic neurons.     

Catecholaminergic properties of cholinergic neurons and synapses in adult rat ciliary ganglion

Parasympathetic neurons of the ciliary ganglion are innervated by preganglionic cholinergic neurons whose cell bodies lie in the brain stem; the ganglion cells in turn provide cholinergic innervation to the intrinsic muscles of the eye. Noradrenergic innervation of the iris is supplied by sympathetic neurons of the superior cervical ganglion. Using immunocytochemical and histochemical techniques, we have examined the ciliary ganglion of adult rats for the expression of cholinergic and noradrenergic properties. As expected, the postganglionic ciliary neurons possessed detectable levels of choline acetyltransferase immunoreactivity (ChAT-IR). Unexpectedly, many ciliary neurons also exhibited immunoreactivity for tyrosine hydroxylase (TH-IR). Some had dopamine beta-hydroxylase-like (DBH-IR) immunoreactivity, but none contained detectable catecholamines, even after treatment with nialamide and L-DOPA. A sparse plexus of fibers exhibiting faint TH-IR was present in the irises of acutely sympathectomized rats. The terminals of preganglionic axons in the ciliary ganglion exhibited not only immunoreactivity for ChAT, but also for TH and contained stores of endogenous catecholamine. Neither ciliary neurons nor their preganglionic innervation accumulated detectable stores of exogenous catecholamines. Rats sympathectomized as neonates by treatment with 6-hydroxydopamine subsequently had a greater proportion of neurons possessing detectable TH-IR in the ciliary ganglion; both the TH-IR perikarya and their axons in the iris were more intensely immunofluorescent. TH-IR was present in the ciliary neuron cell bodies of mouse, guinea pig, and ferret. These species, however, lacked detectable TH-IR or catecholamine stores in preganglionic terminals. These observations indicate that mature, functionally cholinergic neurons from 2 different embryonic origins, postganglionic ciliary neurons derived from the neural crest and preganglionic neurons derived from the neural tube, display several catecholaminergic properties.     

Survival of chick embryo sympathetic neurons in cell culture

Changes in neuronal numbers during the development of the chick embryo paravertebral sympathetic nervous system have been examined using cell culture techniques. Early sympathetic ganglia contain predominantly cells having neuronal phenotypes and these increase in number until embryonic day 9. Subsequently there is a large decrease in the number of neurons and an increase in the population of non-neuronal cells. This in vivo pattern is maintained when the neurons are grown in vitro, where Nerve Growth Factor more readily prevents the death of neurons cultured from 12-day or older embryos than those from earlier stages of development.     

Cholinergic function in cultures of mouse spinal cord neurons

Cholinergic synapses formed in cultures of fetal mouse spinal cord (SC) and superior cervical ganglion (SCG) were studied using intracellular and extracellular stimulation and recording as well as immunohistochemical staining for choline acetyltransferase (ChAT). Dissociated SC neurons and SC explants exhibited cholinergic terminals on SCG and SC neurons as demonstrated by ChAT immunoreactivity. Intracellular recordings showed that cholinergic inputs to SCG neurons were relatively common and that these synaptic inputs were blocked by the nicotinic acetylcholine (ACh) receptor blocker, tubocurarine. A comparison of three preparations indicated that the incidence of cholinergic activity recorded in SCG neurons was significantly higher in co-cultures of SCG with spinal cord ventral horn (VH) neurons grown on a substrate of non-neuronal cells from cerebral cortex, than in co-cultures with VH alone or with SC and dorsal root ganglion cells. Consistency between cholinergic physiology and staining for ChAT-positive terminals on SCG neuronal somata was obtained in cultures of SC explants grown with dissociated SCG. Application of acetylcholine, muscarine, and/or vasoactive intestinal polypeptide (VIP) produced slow excitation of SC neurons. Fast excitatory cholinergic interactions between SC neurons were not observed. Excitatory synaptic interactions between SC neurons were augmented by ACh or muscarine, while inhibitory synaptic interactions were diminished. Both types of synaptic modulation probably were produced by a presynaptic mechanism. Acetylcholine or muscarine affected synaptic interactions between SC neurons in only one-third of the synaptic connections tested, suggesting that the incidence of presynaptically cholinoceptive SC neurons is low in dissociated cell cultures. The experimental results show that a culture system incorporating dissociated fetal mouse SC neurons or explants of SC with sympathetic ganglion neurons expresses both nicotinic and muscarinic cholinergic function.     

Developmental changes in the transmitter properties of sympathetic neurons that innervate the periosteum.

During the development of sweat gland innervation, interactions with the target tissue induce a change from noradrenergic to cholinergic and peptidergic properties. To determine whether the change in neurotransmitter properties that occurs in the sweat gland innervation occurs more generally in sympathetic neurons, we identified a new target of cholinergic sympathetic neurons in rat, the periosteum, which is the connective tissue covering of bone, and characterized the development of periosteal innervation of the sternum. During development, sympathetic axons grow from thoracic sympathetic ganglia along rib periosteum to reach the sternum. All sympathetic axons displayed catecholaminergic properties when they reached the sternum, but these properties subsequently disappeared. Many axons lacked detectable immunoreactivities for vesicular acetylcholine transporter and vasoactive intestinal peptide when they reached the sternum and acquired them after arrival. To determine whether periosteum could direct changes in the neurotransmitter properties of sympathetic neurons that innervate it, we transplanted periosteum to the hairy skin, a noradrenergic sympathetic target. We found that the sympathetic innervation of the transplant underwent a noradrenergic to cholinergic and peptidergic change. These results suggest that periosteum, in addition to sweat glands, regulates the neurotransmitter properties of the sympathetic neurons that innervate it.     

Neuronal stimulation of [3H]thymidine incorporation by primary cultures of highly purified non-neuronal cells

A specific intercellular interaction has been demonstrated between neuronal and non-neuronal cells that appears to increase the rate of non-neuronal cell proliferation. Isolated and recombined primary cultures of both cell types were prepared from 11-day embryonic chick sympathetic ganglia by a method recently developed in this laboratory. When non-dividing neurons were added to an equal number of proliferating non-neuronal cells, the amount of [methyl-³H]thymidine incorporated by these mixed cultures was 230% greater than that incorporated by 99% pure non-neuronal cultures. Removal of all neurons from such non-neuronal cultures by a 48-h preincubation without nerve growth factor resulted in an even greater increase in [³H]thymidine incorporation upon addition of neurons (370%). When increasing numbers of isolated neurons were added to non-neuronal cell cultures, the amount of [³H]thymidine incorporation initially increased in a dose-dependent fashion until it reached a plateau. In contrast, the addition of increasing numbers of non-neuronal cells to a constant number of neurons resulted in a linear increase in [³H]thymidine incorporation. In some cases neurons and non-neuronal cells were not grown in direct physical contact but were only allowed to communicate with one another through the culture medium. Such indirect communication never resulted in a stimulation of [³H]thymidine incorporation. When neurons were added to cultures of embryonic chick fibroblasts, the neurons grew well but did not stimulate [³H]thymidine incorporation by the fibroblasts. These results suggest that embryonic sympathetic neurons selectively stimulate the proliferation of non-neuronal cells derived from the same source.     

Developmental expression of the high affinity choline transporter in cholinergic sympathetic neurons

Choline uptake by the high affinity choline transporter (CHT) is the rate-limiting step in acetylcholine synthesis. Induction of CHT is therefore a critical step in cholinergic differentiation, and we examined the developmental expression of CHT in cholinergic sympathetic neurons that innervate rodent sweat glands. During postnatal development the earliest sympathetic axons in the rear footpads are noradrenergic, containing intense tyrosine hydroxylase immunoreactivity and lacking CHT-immunoreactivity (CHT-IR). By postnatal day 7 (P7) in mouse, and P10 in rat, weak CHT-IR appeared in axons associated with the sweat gland anlagen. CHT staining intensity increased during the following weeks in conjunction with plexus arborization and gland maturation. The pattern of CHT-immunoreactivity (CHT-IR) in the sweat gland innervation was similar to staining for the vesicular acetylcholine transporter and vasoactive intestinal peptide. Immunoblots of tissue from sympathectomized rats confirmed that most of the CHT in footpad was contained in sympathetic neurons. Although CHT expression has been reported in noradrenergic sympathetic neurons of the superior cervical ganglion, these data indicate that in the sympathetic neurons projecting to sweat glands CHT is present at detectable levels only after association with the glands.     

Choline acetyltransferase immunoreactive sympathetic ganglion cells in a teleost,Stephanolepis cirrhifer

The present study showed neurons immunoreactive for choline acetyltransferase (ChAT) in the cranial sympathetic ganglia lying close to the trigeminal-facial nerve complex of the filefish. In these ganglia, less than 1% of ganglion cells were positive for choline acetyltransferase. Choline acetyltransferase-positive neurons were significantly larger than the randomly sampled neurons in this ganglion. The majority of choline acetyltransferase-positive neurons were negative for tyrosine hydroxylase, but many of them were positive for galanin (GAL). Some neurons were positive for both choline acetyltransferase and tyrosine hydroxylase, but these neurons were rarely immunoreactive for dopamine beta hydroxylase, suggesting that they are not adrenergic. In the cranial sympathetic ganglia and the celiac ganglia, many nerve fibers immunoreactive for galanin were seen, and varicose terminals were in contact selectively with neurons negative for both choline acetyltransferase and tyrosine hydroxylase, but not with those positive for choline acetyltransferase or tyrosine hydroxylase. Nerve fibers immunoreactive for choline acetyltransferase were found to be present in contact with the deep layer of chromatophores, which was observed only in the labial region. These results suggest that cholinergic postganglionic neurons are present in the filefish cranial sympathetic ganglia, and that they also contain galanin. As few cholinergic sympathetic neurons express tyrosine hydroxylase and none express dopamine beta hydroxylase, they are unlikely to synthesize noradrenaline or adrenaline.     

Compensatory changes in contralateral sympathetic neurons of the superior cervical ganglion and in their terminals in the pineal gland following unilateral ganglionectomy

The sympathetic noradrenergic neurons of the rat superior cervical ganglia (SCGs) provide the major source of innervation to the pineal gland. The present study sought to determine if this sympathetic innervation can undergo collateral sprouting following partial denervation of the pineal by unilateral removal of the SCG (ganglionectomy), and whether such growth of axon terminals is associated with biochemical changes in the contralateral SCG. In the pineal gland following partial denervation, residual noradrenergic terminals underwent compensatory changes indicative of collateral sprouting, as evidenced by: a rapid reduction in tyrosine hydroxylase (TH) activity and in [3H]norepinephrine (NE) uptake, to about 50% of control by 2 days, which was followed by a gradual but sustained increase to levels of approximately 80% of control by 10 days and a reduction in the intensity and density but not in the distribution of fibers containing NE-induced fluorescence by 2 days, which was followed by a sustained increase. In the contralateral SCG, choline acetyltransferase (CAT) activity, a marker of cholinergic preganglionic terminals, was transiently increased to about 115% of control by 4 days and returned to control levels by 14 days after unilateral ganglionectomy; later, TH activity in noradrenergic cell bodies was gradually increased to about 140% of control by 10 days where it remained for up to 52 days. Unilteral ganglionectomy combined with decentralization of the contralateral SCG by preganglionic nerve cut prevented the compensatory changes in noradrenergic nerve terminals within the pineal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Compensatory elevation of acetylcholine synthesis in vivo by cholinergic neurons surviving partial lesions of the septohippocampal pathway

The present study characterized the effects of partial destruction of the cholinergic septohippocampal pathway on transmitter functions of surviving cholinergic neurons in the hippocampus. Partial and full fimbrial transections were performed, and 3 weeks after lesioning, cholinergic functions were assessed in vivo and in vitro. Hippocampal ChAT activity and the capacity of hippocampal slices to synthesize [3H]ACh in vitro decreased by 35% and 45%, respectively, following partial fimbrial lesions and by 68% and 85%, respectively, following full fimbrial lesions. [3H]ACh release from hippocampal slices in vitro was decreased by 57% and 87%, respectively, following partial and full fimbrial lesions. Partial lesions decreased high-affinity choline uptake into hippocampal synaptosomes by 52%. In contrast to the significant reductions in cholinergic parameters measured in vitro after partial fimbrial lesions, such partial lesions did not significantly alter in vivo measures of hippocampal cholinergic function. Levels of endogenous ACh and choline measured in the hippocampus following partial lesions were similar to that of control values. Also, the hippocampal content of newly synthesized [2H4]ACh and the [2H4]ACh synthesis rate were not significantly different from control values. However, following full fimbrial lesions, in vivo measures of hippocampal cholinergic function were decreased to a degree similar to that observed in vitro. Hippocampal levels of endogenous ACh and [2H4]ACh and the synthesis rate for [2H4]ACh were decreased by 73%, 72%, and 83%, respectively. These results suggest that, following partial destruction of afferent cholinergic fibers that innervate the hippocampal formation, residual cholinergic neurons are able to upregulate their capacity to synthesize and store ACh in vivo, thus compensating for lesion-induced losses of cholinergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)