Cholinergic Innervation Of Hippocampus Research Articles

Epigenetic regulation of microglia and neurons by proinflammatory signaling following adolescent intermittent ethanol (AIE) exposure and in human AUD.

Adolescent alcohol drinking is linked to high rates of adult alcohol problems and alcohol use disorder (AUD). The Neurobiology of Alcohol Drinking in Adulthood (NADIA) consortium adolescent intermittent ethanol (AIE) models adolescent binge drinking, followed by abstinent maturation to adulthood to determine the persistent AIE changes in neurobiology and behavior. AIE increases adult alcohol drinking and preference, increases anxiety and reward seeking, and disrupts sleep and cognition, all risks for AUD. In addition, AIE induces changes in neuroimmune gene expression in neurons and glia that alter neurocircuitry and behavior. HMGB1 is a unique neuroimmune signal released from neurons and glia by ethanol that activates multiple proinflammatory receptors, including Toll-like receptors (TLRs), that spread proinflammatory gene induction. HMGB1 expression is increased by AIE in rat brain and in post-mortem human AUD brain, where it correlates with lifetime alcohol consumption. HMGB1 activation of TLR increase TLR expression. Human AUD brain and rat brain following AIE show increases in multiple TLRs. Brain regional differences in neurotransmitters and cell types impact ethanol responses and neuroimmune gene induction. Microglia are monocyte-like cells that provide trophic and synaptic functions, that ethanol proinflammatory signals sensitize or "prime" during repeated drinking cycles, impacting neurocircuitry. Neurocircuits are differently impacted dependent upon neuronal-glial signaling. Acetylcholine is an anti-inflammatory neurotransmitter. AIE increases HMGB1-TLR4 signaling in forebrain, reducing cholinergic neurons by silencing multiple cholinergic defining genes through upregulation of RE-1 silencing factor (REST), a transcription inhibitor known to regulate neuronal differentiation. HMGB1 REST induction reduces cholinergic neurons in basal forebrain and cholinergic innervation of hippocampus. Adult brain hippocampal neurogenesis is regulated by a neurogenic niche formed from multiple cells. In vivo AIE and in vitro studies find ethanol increases HMGB1-TLR4 signaling and other proinflammatory signaling as well as reducing trophic factors, NGF, and BDNF, coincident with loss of the cholinergic synapse marker vChAT. These changes in gene expression-transcriptomes result in reduced adult neurogenesis. Excitingly, HMGB1 antagonists, anti-inflammatories, and epigenetic modifiers like histone deacetylase inhibitors restore trophic the neurogenesis. These findings suggest anti-inflammatory and epigenetic drugs should be considered for AUD therapy and may provide long-lasting reversal of psychopathology.

Enhanced TrkA signaling impairs basal forebrain-dependent behavior.

Basal forebrain cholinergic neurons (BFCNs) modulate cognitive functions such as attention, learning and memory. The NGF/TrkA pathway plays an important role in the development and function of BFCNs, although two mouse models conditionally deleting TrkA expression in the central nervous system (CNS) have shown contradictory results. To shed light into this discrepancy, we used a mouse model with a gain-of-function in TrkA receptor signaling. Our results indicate that enhanced TrkA signaling did not alter hippocampal cholinergic innervation, general locomotion or anxiety-related behaviors, but it increases ChAT expression, the number of cholinergic neurons at early postnatal stages and, mutant mice showed impaired motor learning and memory functions. These data demonstrate that proper functioning of the cholinergic system in CNS requires a balanced NGF/TrkA signaling.

Early loss of locus coeruleus innervation promotes cognitive and neuropathological changes before amyloid plaque deposition in a transgenic rat model of Alzheimer's disease.

The locus coeruleus (LC) is the main source of noradrenaline (NA) in the mammalian brain and has been found to degenerate during the initial stages of Alzheimer's disease (AD). Recent studies indicate that at late stages of the amyloid pathology, LC-pathological alterations accelerate AD-like pathology progression by interfering with the neuromodulatory and anti-inflammatory properties of NA. However, the impact of LC degeneration at the earliest stages of amyloidosis on the AD-like pathology is not well understood. The LC was lesioned in wild-type and McGill-R-Thy1-APP transgenic (APP tg) rats by administering N-(2-chloroethyl)-N-ethyl-bromo-benzylamine before amyloid plaque deposition. Cognitive deficits and AD-like neuropathological changes were measured after the LC lesion. Four months post-treatment, rats displayed a decrease in brain noradrenergic innervation. The LC lesion in APP tg-treated rats enhanced cognitive deficits and decreased hippocampal cholinergic innervation and neurotrophin expression. In addition, the APP tg-treated rats displayed an increased microglial and astroglial cell number in close vicinity to hippocampal amyloid-beta burdened neurons. The recruited microglia showed cellular alterations indicative of an intermediate activation state. Our results indicate that early LC demise aggravates the early neuroinflammatory process, cognitive impairments, cholinergic deficits and neurotrophin deregulation at the earliest stages of the human-like brain amyloidosis.

Normal cognition in Parkinson's disease may involve hippocampal cholinergic compensation: An exploratory PET imaging study with [18F]-FEOBV

BackgroundSevere cholinergic degeneration is known to occur in Parkinson's disease (PD) and is thought to play a primary role in the cognitive decline associated with this disease. Although cholinergic losses occur in all patients with PD, cognitive performance remains normal for many of them, suggesting compensatory mechanisms in those. ObjectivesThis exploratory study aimed at verifying if normal cognition in PD may involve distinctive features of the brain cholinergic systems. MethodsFollowing extensive neuropsychological screening in 25 patients with PD, 12 were selected and evenly distributed between a cognitively normal (PD-CN) group, and a mild cognitive impairment (PD-MCI) group. Each group was compared with matched healthy volunteers (HV) on standardized cognitive scales (MoCA, PDCRS), and PET imaging with [18F]-FEOBV, a sensitive measurement of brain cholinergic innervation density. Results[18F]-FEOBV uptake reductions were observed in PD-CN as well as in PD-MCI, with the lowest values located in the posterior cortical areas. However, in PD-CN but not in PD-MCI, there was a significant and bilateral increase of [18F]-FEOBV uptake, exclusively located in the hippocampus. Significant correlations were observed between cognitive performance and hippocampal [18F]-FEOBV uptake. ConclusionThese findings suggest a compensatory upregulation of the hippocampal cholinergic innervation in PD-CN, which might underly normal cognitive performances in spite of cortical cholinergic denervation in other regions.

The effect of nerve growth factor on supporting spatial memory depends upon hippocampal cholinergic innervation

Nerve growth factor (NGF) gene therapy has been used in clinical trials of Alzheimer’s disease. Understanding the underlying mechanisms of how NGF influences memory may help develop new strategies for treatment. Both NGF and the cholinergic system play important roles in learning and memory. NGF is essential for maintaining cholinergic innervation of the hippocampus, but it is unclear whether the supportive effect of NGF on learning and memory is specifically dependent upon intact hippocampal cholinergic innervation. Here we characterize the behavior and hippocampal measurements of volume, neurogenesis, long-term potentiation, and cholinergic innervation, in brain-specific Ngf-deficient mice. Our results show that knockout mice exhibit increased anxiety, impaired spatial learning and memory, decreased adult hippocampal volume, neurogenesis, short-term potentiation, and cholinergic innervation. Overexpression of Ngf in the hippocampus of Ngf gene knockout mice rescued spatial memory and partially restored cholinergic innervations, but not anxiety. Selective depletion of hippocampal cholinergic innervation resulted in impaired spatial memory. However, Ngf overexpression in the hippocampus failed to rescue spatial memory in mice with hippocampal-selective cholinergic fiber depletion. In conclusion, we demonstrate the impact of Ngf deficiency in the brain and provide evidence that the effect of NGF on spatial memory is reliant on intact cholinergic innervations in the hippocampus. These results suggest that adequate cholinergic targeting may be a critical requirement for successful use of NGF gene therapy of Alzheimer’s disease.

M1 muscarinic allosteric modulators slow prion neurodegeneration and restore memory loss.

The current frontline symptomatic treatment for Alzheimer’s disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR–selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD.

Effects of Maternal Choline Supplementation on the Septohippocampal Cholinergic System in the Ts65Dn Mouse Model of Down Syndrome.

Down syndrome (DS), caused by trisomy of chromosome 21, is marked by intellectual disability (ID) and early onset of Alzheimer's disease (AD) neuropathology including hippocampal cholinergic projection system degeneration. Here we determined the effects of age and maternal choline supplementation (MCS) on hippocampal cholinergic deficits in Ts65Dn mice compared to 2N mice sacrificed at 6-8 and 14-18 months of age. Ts65Dn mice and disomic (2N) littermates sacrificed at ages 6-8 and 14-18 mos were used for an aging study and Ts65Dn and 2N mice derived from Ts65Dn dams were maintained on either a choline-supplemented or a choline-controlled diet (conception to weaning) and examined at 14-18 mos for MCS studies. In the latter, mice were behaviorally tested on the radial arm Morris water maze (RAWM) and hippocampal tissue was examined for intensity of choline acetyltransferase (ChAT) immunoreactivity. Hippocampal ChAT activity was evaluated in a separate cohort. ChAT-positive fiber innervation was significantly higher in the hippocampus and dentate gyrus in Ts65Dn mice compared with 2N mice, independent of age or maternal diet. Similarly, hippocampal ChAT activity was significantly elevated in Ts65Dn mice compared to 2N mice, independent of maternal diet. A significant increase with age was seen in hippocampal cholinergic innervation of 2N mice, but not Ts65Dn mice. Degree of ChAT intensity correlated negatively with spatial memory ability in unsupplemented 2N and Ts65Dn mice, but positively in MCS 2N mice. The increased innervation produced by MCS appears to improve hippocampal function, making this a therapy that may be exploited for future translational approaches in human DS.

Hippocampal acetylcholine depletion has no effect on anxiety, spatial novelty preference, or differential reward for low rates of responding (DRL) performance in rats.

We investigated the role of the septo-hippocampal cholinergic projection in anxiety, spatial novelty preference, and differential reward for low rates of responding (DRL) performance. Cholinergic neurons of the rat medial septum (MS) and the vertical limb of the diagonal band of Broca (VDB) were lesioned using the selective immunotoxin, 192 IgG-saporin. Rats were then tested on several behavioral tests previously shown to be sensitive to either (a) hippocampal lesions or (b) nonselective MS/VDB lesions which target both cholinergic and γ-aminobutyric acid (GABA)-ergic projections, or both. Saporin lesions substantially reduced hippocampal cholinergic innervation, resulting in an absence of acetyl cholinesterase staining and markedly reduced choline acetyltransferase activity (mean reduction: 80 ± 5%; range: 50–97%). However, the saporin-lesioned rats did not differ from control rats in any of the behavioral tests. Thus we found no evidence from these lesion studies that the septo-hippocampal cholinergic projection plays an essential role in anxiety, spatial novelty preference, or DRL.

Episodic memory: neurological and neuromediator mechanisms

Significant advances in understanding of neurological and neuromediator mechanisms of memory along with the causes of memory decline in aging were achieved recently. Functioning of episodic memory system needs considerable energetic and material (neuromediator, protein) resources, and appears to be highly consuming for the body. Therefore, physiological mechanisms inhibiting episodic memory system exist in order to distribute resources for other brain functions. Primary engram is recorded by hippocampal structures, which maintain reciprocal connectivity with neocortex zones involved in synchronized activity. Cholinergic innervation of hippocampus and primary zones of neocortex stabilizes process of primary engram formation with consequent transformation of synapses in hippocampus structures. At the next stage hippocampus transfers the engram to neocortex, where the information is processed by associating with previous knowledge and is fixed by slowly developing synaptogenesis and axonal growth. Noradrenergic innervation is an essential part of neuroplastic processes. Possible causes of memory decline in aging include powerful influences of mature prefrontal cortex, inhibiting the hippocampal activity; insufficient stimulation of hippocampal memory system by novel events with consequent decline of neurogenesis and neuroplasticity; disturbances in mechanisms of phasic release and clearance of neuromediators.

Coexpression of glutamate vesicular transporter (VGLUT1) and choline acetyltransferase (ChAT) proteins in fetal rat hippocampal neurons in culture

A very small population of choline acetyltransferase (ChAT) immunoreactive cells is observed in all layers of the adult hippocampus. This is the intrinsic source of the hippocampal cholinergic innervation, in addition to the well-established septo-hippocampal cholinergic projection. This study aimed at quantifying and identifying the origin of this small population of ChAT-immunoreactive cells in the hippocampus at early developmental stages, by culturing the fetal hippocampal neurons in serum-free culture and on a patternable, synthetic silane substrate N-1 [3-(trimethoxysilyl) propyl] diethylenetriamine. Using this method, a large proportion of glutamatergic (glutamate vesicular transporter, VGLUT1-immunoreactive) neurons, a small fraction of GABAergic (GABA-immunoreactive) neurons, and a large proportion of cholinergic (ChAT-immunoreactive) neurons were observed in the culture. Interestingly, most of the glutamatergic neurons that expressed glutamate vesicular transporter (VGLUT1) also co-expressed ChAT proteins. On the contrary, when the cultures were double-stained with GABA and ChAT, colocalization was not observed. Neonatal and adult rat hippocampal neurons were also cultured to verify whether these more mature neurons also co-express VGLUT1 and ChAT proteins in culture. Colocalization of VGLUT1 and ChAT in these relatively more mature neurons was not observed. One possible explanation for this observation is that the neurons have the ability to synthesize multiple neurotransmitters at a very early stage of development and then with time follows a complex, combinatorial strategy of electrochemical coding to determine their final fate.

Sympathetic sprouting in visual cortex stimulated by cholinergic denervation rescues expression of two forms of long-term depression at layer 2/3 synapses

Cholinergic innervation of hippocampus and cortex is required for some forms of learning and memory. Several reports have shown that activation of muscarinic m1 receptors induces a long-term depression (mLTD) at glutamate synapses in hippocampus and in several areas of cortex, including perirhinal and visual cortices. This plasticity likely contributes to cognitive function dependent upon the cholinergic system. In rodent models, degeneration of hippocampal cholinergic innervation following lesion of the medial septum stimulates sprouting of adrenergic sympathetic axons, originating from the superior cervical ganglia (SCG), into denervated hippocampal subfields. We previously reported that this adrenergic sympathetic sprouting occurs simultaneously with a reappearance of cholinergic fibers in hippocampus and rescue of mLTD at CA3-CA1 synapses. Because cholinergic neurons throughout basal forebrain degenerate in aging and Alzheimer's disease, it is critical to determine if this compensatory sprouting occurs in other regions impacted by cholinergic cell loss. To this end, we investigated whether lesion of the nucleus basalis magnocellularis (NbM) to cholinergically denervate cortex stimulates adrenergic sympathetic sprouting and the accompanying increase in cholinergic innervation. Further, we assessed whether the presence of sprouting positively correlates with the ability of glutamate synapses in acute visual cortex slices to express mLTD and low frequency stimulation induced LTD (LFS LTD), another cholinergic dependent form of plasticity in visual cortex. We found that both mLTD and LFS LTD are absent in animals when NbM lesion is combined with bilateral removal of the SCG to prevent possible compensatory sprouting. In contrast, when the SCG remain intact to permit sprouting in animals with NbM lesion, cholinergic fiber density is increased concurrently with adrenergic sympathetic sprouting, and mLTD and LFS LTD are preserved. Our findings suggest that autonomic compensation for central cholinergic degeneration is not specific to hippocampus, but is a general repair mechanism occurring in other brain regions important for normal cognitive function.

Developmental forebrain cholinergic lesion and environmental enrichment: Behaviour, CA1 cytoarchitecture and neurogenesis

Intraventricular injections of 192 IgG saporin in 7-day-old rat severely reduced hippocampal cholinergic innervation as reflected by both decreased acetylcholinesterase staining and immunoreactivity for the p75 neurotrophin receptor. It was determined if this altered the effects of environmental enrichment on spatial learning, hippocampal CA1 cell cytoarchitecture as reflected by the Golgi stain, and neurogenesis in the dentate gyrus as indicated by doublecortin immunoreactivity. At weaning, lesioned and control rats were either group housed in large, environmentally enriched cages or housed two per standard cage for 42 days. When subsequently assessed with a working-memory spatial navigation task, both lesioned and control rats showed enhanced learning as a result of enrichment. Quantitative analysis of Golgi stained sections indicated that enrichment did not affect CA1 dendritic branching, total dendritic length or dendritic spine density. However, the lesion reduced the number of apical branches, spine density on intermediate to distal apical dendrites, and the length of basal branches. It also reduced the number of doublecortin immunoreactive neurons in the dentate gyrus and appeared to prevent their increase due to environmental enrichment. It is concluded that developmental cholinergic lesioning does not attenuate neurobehavioral plasticity, at least as reflected by the behavioral consequences of enrichment. It does, however, attenuate neurogenesis in the dentate gyrus, like adult-inflicted cholinergic lesions. As previously found for cortical neurons, it also reduces CA1 pyramidal cell dendritic complexity and spine density in adulthood. The results have implications for the loss of synapses that occurs in both developmental and aging-related brain disorders involving cholinergic dysfunction.

Chronic brain cytochrome oxidase inhibition selectively alters hippocampal cholinergic innervation and impairs memory: Prevention by ladostigil

A 25–35% reduction of brain cytochrome oxidase (COx) activity found in Alzheimer's disease (AD) could contribute to neuronal dysfunction and cognitive impairment. The present study replicated the reduction in brain COx activity in rats by administering sodium azide (NaN 3) for 4 weeks via Alzet minipumps at the rate of 1 mg/kg/h, and determined its effect on hippocampal cholinergic transmission, spatial and episodic memory. NaN 3 caused a selective reduction in choline acetyltransferase (ChAT) immunoreactivity in the diagonal band, a major source of cholinergic input to the hippocampus and cingulate cortex, without altering the number of cholinergic neurons. NaN 3 also induced a significant increase in vesicular acetylcholine transporter (VAChT)-immunoreactive varicosities, GAP-43 in the subgranular layer and of transferrin receptors (TfR) in the hilus of the dentate gyrus. These neurochemical changes were associated with impairment in spatial learning in the Morris water maze and in episodic memory in the object recognition test. Chronic treatment with ladostigil, a novel cholinesterase and monoamine oxidase inhibitor, prevented the decrease in ChAT in the diagonal band, the compensatory increase in synaptic plasticity and TfR and the memory deficits without restoring COx activity. Ladostigil had no significant effect on ChAT activity, synaptic plasticity or TfR in control rats. Ladostigil may have a beneficial effect on cognitive deficits in AD patients that have a reduction in cortical COx activity and cholinergic hypofunction.

Mechanism‐Based Approaches for the Reversal of Drug Neurobehavioral Teratogenicity

Understanding the mechanism of neurobehavioral teratogenicity is the primary prerequisite for reversal of the defect. Progress in such studies can be best achieved if the investigation focuses on behaviors related to a specific brain region and innervation. Our model focused on teratogen-induced deficits in hippocampus-related eight-arm and Morris maze behaviors. Different "cholinergic" teratogens, mainly heroin, induced both pre- and postsynaptic hyperactivity in the hippocampal cholinergic innervation that terminated in desensitization of Protein Kinase C (PKC) isoforms to cholinergic receptor stimulation. Understanding this mechanism enabled its reversal with a pharmacological therapy-nicotine infusion. Studies by others provided similar findings by targeting the deficits respective to the model investigated. Consistently, destruction of the A10-septal dopaminergic pathways that downregulate the septohippocampal cholinergic innervation ameliorated maze performance. Grafting of embryonic differentiated cholinergic cells or neural progenitors similarly reversed the biochemical/molecular alterations and the resulting deficits. Reversal therapies offer a model for the understanding of neurobehavioral teratogenicity and, clinically, offer a model for potential treatment of these deficits. Whereas neural progenitor grafting appears to be the most effective treatment, pharmacological reversal with nicotine infusion seems to possess the most feasible and immediate therapy for neurobehavioral birth defects produced by various teratogens, including drugs. This is true even though the effect of pharmacological therapies is diffuse, affecting multiple areas of the brain. "Everybody is talking about the weather but nobody does anything about it." (Mark Twain).

Postnatal development of the cholinergic innervation in the dorsal hippocampus of rat: Quantitative light and electron microscopic immunocytochemical study

Choline acetyltransferase (ChAT) immunocytochemistry was used to examine the distribution and ultrastructural features of the acetylcholine (ACh) innervation in the dorsal hippocampus of postnatal rat. The length of ChAT-immunostained axons was measured and the number of ChAT-immunostained varicosities counted, in each layer of CA1, CA3, and dentate gyrus, at postnatal ages P8, P16, and P32. At P8, an elaborate network of varicose ChAT-immunostained axons was already visible. At P16, the laminar distribution of this network resembled that in the adult, but adult densities were reached only by P32. Between P8 and P32, the mean densities for the three regions increased from 8.4 to 14 meters of axons and 2.3 to 5.7 million varicosities per cubic millimeter of tissue. At the three postnatal ages, the ultrastructural features of ChAT-immunostained axon varicosities from the strata pyramidale and radiatum of CA1 were similar between layers and comparable to those in adult, except for an increasing frequency of mitochondria (up to 41% at P32). The proportion of these profiles displaying a synaptic junction was equally low at all ages, indicating an average synaptic incidence of 7% for whole varicosities, as previously found in adult. The observed junctions were small, usually symmetrical, and made mostly with dendritic branches. These results demonstrate the precocious and rapid maturation of the hippocampal cholinergic innervation and reveal its largely asynaptic nature as soon as it is formed. They emphasize the remarkable growth capacities of individual ACh neurons and substantiate a role for diffuse transmission by ACh during hippocampal development.

Convergent Effects on Cell Signaling Mechanisms Mediate the Actions of Different Neurobehavioral Teratogens: Alterations in Cholinergic Regulation of Protein Kinase C in Chick and Avian Models

Although the actions of heroin on central nervous system (CNS) development are mediated through opioid receptors, the net effects converge on dysfunction of cholinergic systems. We explored the mechanisms underlying neurobehavioral deficits in mouse and avian (chick, Cayuga duck) models. In mice, prenatal heroin exposure (10 mg/kg on gestation days 9-18) elicited deficits in behaviors related to hippocampal cholinergic innervation, characterized by concomitant pre- and postsynaptic hyperactivity, but ending in a reduction of basal levels of protein kinase C (PKC) isoforms betaII and gamma and their desensitization to cholinergic receptor-induced activation. PKCalpha, which is not involved in the behaviors studied, was unaffected. Because mammalian models possess inherent confounding factors from maternal effects, we conducted parallel studies using avian embryos, evaluating hyperstriatal nucleus (intermedial part of the hyperstriatum ventrale, IMHV)-related, filial imprinting behavior. Heroin injection to the eggs (20 mg/kg) on incubation days 0 and 5 diminished the post-hatch imprinting ability and reduced PKCg and bII content in the IMHV membrane fraction. Two otherwise unrelated agents that converge on cholinergic systems, chlorpyrifos and nicotine, elicited the same spectrum of effects on PKC isoforms and imprinting but had more robust actions. Pharmacological characterization also excluded direct effects of opioid receptors on the expression of imprinting; instead, it indicated participation of serotonergic innervation. The avian models can provide rapid screening of neuroteratogens, exploration of common mechanisms of behavioral disruption, and the potential design of therapies to reverse neurobehavioral deficits.

Reduction in hippocampal cholinergic innervation is unrelated to recognition memory impairment in aged rhesus monkeys.

Alterations in the basal forebrain cholinergic system have been widely studied in brain aging and Alzheimer's disease, but the magnitude of decline and relationship to cognitive impairment are still a matter of debate. The rhesus monkey (Macaca mulatta) provides a compelling model to study age-related memory decline, as the pattern of impairment closely parallels that observed in humans. Here, we used antibodies against the vesicular acetylcholine transporter and a new stereological technique to estimate total cholinergic fiber length in hippocampal subregions of behaviorally characterized young and aged rhesus monkeys. The analysis revealed an age-related decline in the length of cholinergic fibers of 22%, which was similar across the hippocampal subregions studied (dentate gyrus granule cell and molecular layers, CA2/3-hilus, and CA1), and across the rostral-caudal extent of the hippocampus. This effect, however, was unrelated to performance on the delayed nonmatching-to-sample task, a test of recognition memory sensitive to hippocampal system dysfunction and cognitive aging in monkeys. These findings indicate that a decline in cholinergic input fails to account for the influence of normal aging on memory supported by the primate hippocampal region.

A decrease in size and number of basal forebrain cholinergic neurons is paralleled by diminished hippocampal cholinergic innervation in mice lacking leukocyte common antigen-related protein tyrosine phosphatase activity

The leukocyte common antigen-related (LAR) receptor, composed of an extracellular region with three immunoglobulin-like and eight fibronectin type III-like domains, and a cytoplasmic region containing two protein tyrosine phosphatase domains, is thought to play a role in axonal outgrowth and guidance during neural development. LAR mutant mice were generated completely lacking the two cytoplasmic protein tyrosine phosphatase domains, resulting in the loss of ability to bind intracellular associating proteins, but (may be) still containing the ability to perform extracellular functions. A reduction in size of basal forebrain cholinergic neurons and diminished hippocampal innervation reported for knockout mice that contain a leaky gene trap inserted into the 5′ part of the LAR gene [Yeo T. T. et al. (1997) J. Neurosci. Res. 47, 348–360] warranted a computer-assisted quantitative image analysis throughout the basal forebrain and hippocampus of our LAR mutant mice. The total number, longest diameter and cell body area were calculated for the choline acetyltransferase-positive neurons in the medial septum and vertical diagonal band, and optical density measurements were performed to determine the extent of acetyl cholinesterase-positive fibre innervation of the different layers in the dentate gyrus. In LAR mutant mice, the number of cholinergic cells was significantly reduced (∼25%) in the vertical diagonal band. Also, the cross-sectional area of the cholinergic neurons in the medial septum and vertical diagonal band was reduced (5%). These findings were paralleled by a diminished cholinergic innervation of the supragranular (18%) and molecular (4%) layers of the dentate gyrus. Thus, LAR protein tyrosine phosphatase activity appears crucial for size, number and target projection of basal forebrain cholinergic neurons, further strengthening a role for LAR in CNS development.

Characterization of opioid receptor types modulating acetylcholine release in septal regions of the rat brain.

Presynaptic opioid receptors of the delta- and mu-types have been shown to inhibit the release of acetylcholine (ACh) in the rat striatum and hippocampus, respectively, but it is unknown whether opioid receptors modulate the release of ACh also in the region of origin of the hippocampal cholinergic innervation, the septum. To answer this question, slices (350 microm) of the medial septal area and of the diagonal band of Broca, as well as (for comparison) of the hippocampus, were prepared from adult male Wistar rats. The slices were incubated with [3H]choline, superfused in the presence of hemicholinium-3 (10 microM) and stimulated twice (S1, S2) by electrical fields (360 pulses, 3 Hz, 2 ms, 60 mA); opioid receptor agonists were present during S2. The preferential mu-agonist [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) inhibited the evoked ACh release by maximally about 40% in hippocampal slices and acted even more strongly in the medial septal area, or the diagonal band of Broca (about 60% or 75% maximal inhibition, respectively). These effects were reduced or abolished by the preferential mu-antagonist naloxone, which showed no effects when given alone. Using naloxone in the presence of a cocktail of peptidase inhibitors, no evidence for an endogenous tone of opioid peptides was found in the medial septal area, diagonal band of Broca or the hippocampus. Using the preferential delta-agonist [D-Pen2, D-Pen5]enkephalin (DPDPE) and the delta-antagonist naltrindole, a delta-opioid receptor inhibiting evoked ACh release was clearly detectable both in the medial septal area and the diagonal band of Broca, but not in the hippocampus, whereas the preferential kappa-agonist trans-3,4-dichloro-N-methyl-N-[2(1-pyrrolidinyl)cyclo-hexyl] benzeneacetamide (U50,488H) had only weak or no effects. In addition to the functional experiments, double in-situ hybridization studies were performed, in which cells containing mRNA for choline acetyltransferase (ChAT) were labeled by an antibody-linked enzymatic staining procedure, whereas mRNAs for mu- or delta-opioid receptors were detected with radioactive probes. These experiments revealed that in the septal region mainly mu-opioid receptors were expressed by neurons positive for ChAT mRNA, whereas in the rat striatum the expression of delta-opioid receptors prevailed in those neurons. We conclude that in the septal area of the rat brain, in contrast to the rat striatum and hippocampus, both presynaptic mu- and delta-opioid receptors modulate the evoked release of ACh. Whether presynaptic mu- and delta-opioid receptors occur on the same or on different septal cells or axon terminals remains to be clarified.

GM1 and Piracetam Do Not Revert the Alcohol-Induced Depletion of Cholinergic Fibers in the Hippocampal Formation of the Rat

BRANDÃO, F., A. RIBEIRO-DA-SILVA AND A. CADETE-LEITE. GM1 and piracetam do not revert the alcohol-induced depletion of cholinergic fibers in the hippocampal formation of the rat. ALCOHOL 19(1) 65–74, 1999.—Chronic alcohol consumption causes a depletion of the cholinergic fiber network in the rat hippocampal formation, which is not ameliorated by alcohol withdrawal. Following withdrawal from alcohol, there is a further loss of intrinsic hippocampal cholinergic neurons. In this study, we investigated whether treatment with putative neuroprotective agents during the entire withdrawal period would have beneficial effects upon the hippocampal cholinergic innervation. Adult male rats were alcohol-fed for 6 months and subsequently withdrawn from alcohol for 6 months. Some animals were treated with either ganglioside GM1 (35 mg/kg body weight s.c.), vehicle (saline s.c.), or piracetam (800 mg/kg body weight p.o.) for the entire withdrawal period. Choline acetyltransferase (ChAT) immunoreactive (IR) fibers and neurons were analyzed quantitatively in all four animal groups. There were no significant differences in the density of the ChAT-IR hippocampal fiber network when the pure withdrawal and withdrawal + vehicle groups were compared to the withdrawal + GM1 or withdrawal + piracetam groups. In contrast, the number of ChAT-IR interneurons in the hippocampal formation was higher in the withdrawal + GM1 or withdrawal + piracetam groups than in the pure withdrawal and withdrawal + vehicle groups. These results indicate that, in the doses used, neither neuroprotective agent had an effect upon the extrinsic cholinergic innervation, but they had a beneficial effect upon the hippocampal intrinsic cholinergic system.