Narcoleptic orexin receptor knockout mice express enhanced cholinergic properties in laterodorsal tegmental neurons.

Pharmacological studies of narcoleptic canines indicate that exaggerated pontine cholinergic transmission promotes cataplexy. As disruption of orexin (hypocretin) signaling is a primary defect in narcolepsy with cataplexy, we investigated whether markers of cholinergic synaptic transmission might be altered in mice constitutively lacking orexin receptors (double receptor knockout; DKO). mRNA for Choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and the high-affinity choline transporter (CHT1) but not acetylcholinesterase (AChE) was significantly higher in samples from DKO than wild-type (WT) mice. This was region-specific; levels were elevated in samples from the laterodorsal tegmental nucleus (LDT) and the fifth motor nucleus (Mo5) but not in whole brainstem samples. Consistent with region-specific changes, we were unable to detect significant differences in Western blots for ChAT and CHT1 in isolates from brainstem, thalamus and cortex or in ChAT enzymatic activity in the pons. However, using ChAT immunocytochemistry, we found that while the number of cholinergic neurons in the LDT and Mo5 were not different, the intensity of somatic ChAT immunostaining was significantly greater in the LDT, but not Mo5, from DKO than from WT mice. We also found that ChAT activity was significantly reduced in cortical samples from DKO compared with WT mice. Collectively, these findings suggest that the orexins can regulate neurotransmitter expression and that the constitutive absence of orexin signaling results in an up-regulation of the machinery necessary for cholinergic neurotransmission in a mesopontine population of neurons that have been associated with both normal rapid eye movement sleep and cataplexy.

Reduction of cerebrospinal fluid amyloid beta after systemic administration of M1 muscarinic agonists.

Overproduction of the peptide amyloid beta (A beta) is a critical event in Alzheimer's disease (AD). Systemic administration of 3 M1-selective muscarinic agonists, AF102B, AF150S and AF267B, decreased cerebrospinal fluid (CSF) A beta concentrations; levels of CSF secreted beta-APP were not significantly altered. Rabbits treated for 5 days with s.c. injections of each drug (2 mg/kg/day) had levels of CSF A beta which were between 55 and 71% of control for A beta 1-40 and between 59 and 84% of control for A beta 1--42.

Cortical cholinergic denervation elicits vascular A beta deposition.

Selective destruction of the cholinergic nucleus basalis magnocellularis (nbm) in the rabbit by the p75 neurotrophin receptor (NTR) immunoglobulin G (IgG) complexed to the toxin saporin leads to the deposition of amyloid-beta (A beta) in and around cerebral blood vessels. In some instances, the perivascular A beta resemble the diffuse deposits observed in Alzheimer's disease (AD). We propose that cortical cholinergic deprivation results, among other perturbations, in the loss of vasodilation mediated by acetylcholine. In addition to a dysfunctional cerebral blood flow, alterations in vascular chemistry affecting endothelial and smooth muscle cells may result in cerebral hypoperfusion and a breached blood-brain barrier (BBB). The selective removal of the rabbit nbm and A beta accumulation may serve as an important nontransgenic, and more physiological, model for the testing of pharmacological and immunological agents designed to control the deposition and the deleterious effects of A beta in AD.

Cholinergic deafferentation of the rabbit cortex: a new animal model of Abeta deposition.

Brain deposition of the amyloid beta-peptide (Abeta) is a critical step in the pathogenesis of Alzheimer's disease (AD) and human cerebral amyloid angiopathy (CAA). A small fraction of AD and CAA cases are caused by gene mutations leading to increased production and deposition of Abeta, but for the majority, there is no known direct genetic cause. We have hypothesized that Abeta deposition in these sporadic cases occurs as a result of cortical cholinergic deafferentation. Here we show that cortical cholinergic deafferentation, induced in rabbits by a selective immunotoxin, leads to Abeta deposition in cerebral blood vessels and perivascular neuropil. Biochemical measurements confirmed that lesioned animals had 2.5- and 8-fold elevations of cortical Abeta40 and Abeta42, respectively. Cholinergic deafferentation may be one factor that can contribute to Abeta deposition.

Branched-chain amino acid-induced hippocampal norepinephrine release is antagonized by picrotoxin: evidence for a central mode of action.

Previous studies indicated that administration of a 1:1:1 mixture of the branched-chain amino acids leucine, isoleucine, and valine (BCAA) decreased the response to pain. The present study investigates the effects of BCAA on release of norepinephrine (NE) from isolated hippocampal brain slices. BCAA evoked 3H-NE release in a concentration-dependent manner. This effect was antagonized by the gamma aminobutyric acid (GABA) receptor antagonist picrotoxin, again in a concentration-dependent manner, suggesting that the effect may be mediated via a GABA receptor. Given the role of NE and of GABA receptors in the central response to pain, it is possible that the BCAA may exert their antinociceptive properties through activation of GABA receptors.

Lesion of septal-hippocampal neurons with 192 IgG-saporin alters function of M1 muscarinic receptors.

Cholinergic neurons projecting from the medial septum to the hippocampus were lesioned with the selective neurotoxin 192 IgG-saporin. Injection of 300 ng of 192 IgG-saporin into the medial septum produced a 60% decrease in choline acetyltransferase activity. M1 muscarinic receptor function was examined by measuring enhancement of evoked release of norepinephrine from rat hippocampal slices by the M1 selective agonist McN-A-343. In hippocampal slices from rats which were lesioned with 192-saporin, the response to McN-A-343 was reduced compared to sham-operated controls. Pirenzepine binding demonstrated no change in M1 receptor number or affinity. However, the curve for displacement of pirenzepine by the muscarinic agonist oxotremorine-M was shifted to the right in hippocampal tissue from lesioned rats. This shift was identical to that produced by addition of the non-hydrolyzable GTP analogue GppNHp, which uncouples the M1 muscarinic receptor/G-protein complex. These results suggest that lesion of septal-hippocampal cholinergic inputs causes uncoupling of the M1 muscarinic receptor, decreasing responsiveness to stimulation. These findings are similar to reports of decreased M1 muscarinic receptor coupling to G-proteins and loss of function in Alzheimer's disease. The 192 IgG-saporin lesion may provide a viable animal model in which to study uncoupling of G-proteins and M1 muscarinic receptors.

Purification of human fetal hippocampal neurons by flow cytometry for transplantation.

We have established primary cultures, highly enriched in neurons, from the hippocampus of human fetal brains at 20-23 gestational weeks. More than 80% of cells were viable when seeded. Neurons were isolated from primary cultures by flow cytometry to a high degree of purity, as demonstrated by immunocytochemical staining. FACS scanning analysis using a DNA-staining dye showed that hippocampal neurons did not divide in culture. To demonstrate that FACS-sorted neurons can be transplanted and integrated into the host brain, neuron-enriched primary culture from human fetal striatum was infected with a viral-mediated vector containing a reporter gene, beta-galactosidase. Striatal neurons were subsequently purified by flow cytometry and transplanted into the striatum of rats. Following transplantation, the rat brains were processed for beta-galactosidase histochemistry and electron microscopy. Beta-galactosidase expression indicates that transplanted human neurons survived in the host and were metabolically active. The transplanted neurons received synaptic inputs, as judged from the presence of presynaptic terminals on their surface. Our study demonstrates connectivity between transplanted human fetal primary neurons and host tissue at the ultrastructural level. Our results support the feasibility of ultimately transplanting neurons into humans as a possible treatment for recovery of the nervous system (e.g., neurodegenerative diseases).

alpha 2A subtype of presynaptic alpha 2-adrenoceptors modulates the release of [3H]-noradrenaline from rat spinal cord.

The modulation of noradrenaline (NA) release from rat spinal cord slices was investigated and the subtype of presynaptic alpha 2-adrenoceptors involved in the negative feedback modulation was characterized using in vitro perfusion experiments. Rat spinal cord slices were loaded with [3H]NA and the release of radioactivity at rest and in response to field stimulation was determined. The alpha 2-adrenoceptor agonists, clonidine and dexmedetomidine inhibited the stimulation-evoked release of NA from spinal cord slices, whereas alpha 2-adrenoceptor antagonists yohimbine and CH-38083 (7,8-(methylenedioxy)-14-alpha-hydroxyalloberbane HCI), enhanced it. ARC 239, a selective alpha 2B-antagonist, had no effect on the release. In contrast, BRL 44408 a selective alpha 2A-antagonist increased the release of NA. Our results indicate that the negative feedback modulation of NA release from noradrenergic fibres in the spinal cord is mediated via alpha 2A subtype of alpha 2-adrenoceptors.

Modulation of hippocampal norepinephrine release by cholinergic agonists is altered by AF64A lesion.

The effect of lesioning hippocampal cholinergic neurons with the neurotoxin AF64A on the ability of cholinergic agonists to modulate stimulation-induced release of 3H-norepinephrine (NE) from rat hippocampal slices was studied. Rats received intracerebroventricular injections of either AF64A (ethylcholine mustard aziridinium, 2 nmol) or vehicle (sham operated). Six weeks after treatment, release of 3H-NE evoked by electrical stimulation (2 Hz, 2 min) in the presence or absence of cholinergic agonists and/or antagonists was measured. Activation of M2 receptors with oxotremorine (in the presence of the M1 antagonist pirenzepine) caused a small inhibition of NE release, which was abolished in hippocampi from AF64A-treated rats. The Kd for high-affinity binding of the selective M2 ligand [3H] AF-DX 384 was increased 10-fold in lesioned tissues. The M1 selective agonist McN-A-343 produced a significant enhancement of NE release, which was unchanged by AF64A lesion. Binding studies with [3H] pirenzepine showed no change in the affinity or number of M1 receptors. Nicotine also caused a significant enhancement of evoked NE release, but this effect was markedly reduced in tissues from AF64A-treated rats. AF64A treatment caused a twofold decrease in the number of [3H] nicotine binding sites. This study suggests that long-term lesion of hippocampal cholinergic neurons with AF64A alters the function of postsynaptic muscarinic M2 and nicotinic cholinergic receptors that modulate the release of NE in the hippocampus.

Lesion with the neurotoxin AF64A alters hippocampal cholinergic receptor function.

The effect of selective lesion of cholinergic inputs to the hippocampus on the function of hippocampal cholinergic receptors was examined. Hippocampal cholinergic neurons were lesioned in the rat by administration of the selective cholinergic neurotoxin AF64A (ethylcholine mustard aziridinium). Cholinergic receptor function was examined by assessing the ability of cholinergic agonists and antagonists to modulate the evoked release of radiolabelled acetylcholine (ACh) from hippocampal slices. Nicotine enhanced release, with a bell-shaped dose-response curve. The dose-response curve and EC50 for nicotine was shifted 10-fold to the left in lesioned rats, suggesting an increased sensitivity to nicotine. However, there were no differences in either the number of affinity of nicotinic receptors as determined with binding studies. The muscarinic agonist oxotremorine inhibited the evoked release of ACh in control tissues, but had much less effect in AF64A-lesioned tissues. Binding to the M1 receptor subtype was not changed. However, the Kd for binding to the high affinity subtype of the M2 receptor was increased 10-fold, suggesting that the receptor has become less sensitive to stimulation. Loss of M2 function may allow an increase in the effect of stimulating nicotinic receptors that modulate ACh release.

Simultaneous single-cell recording and microdialysis within the same brain site in freely behaving rats: a novel neurobiological method.

We present a method for performing intracerebral microdialysis in freely behaving rats while recording the firing of neurons within the dialysis site. Studying hippocampal theta cells and complex-spike cells with this technique, it has been found that: (1) when the microdialysis fluid contained only artificial cerebrospinal fluid, both types of neurons displayed normal electrical activity, (2) the simultaneous single-cell recording/microdialysis procedure could be readily performed for as long as 3 days, and (3) inclusion of drugs into the microdialysis fluid, at appropriate concentrations, caused clear changes in firing pattern. For example, microdialysis with 1% lidocaine completely abolished, whereas that with 50 mM K+ markedly increased, the neuronal electrical activity. These cellular changes developed without apparent EEG or behavioral manifestations and were reversible. In some of the experiments, the extracellular concentrations of glutamate and aspartate in the recording/dialysis site were also measured. The described method allows the extracellular environment of recorded brain cells to be manipulated by drugs delivered through the microdialysis probe and simultaneously allows determination of the neurochemical composition of that environment over a remarkably long period of time and in intact, physiologically functioning, neural network. Such studies will provide new insights into the molecular basis of neuronal activity in the brain in the context of behavior, including learning.

Phenytoin protects against hypoxia-induced death of cultured hippocampal neurons.

The neuroprotective actions of the anticonvulsant phenytoin (diphenylhydantoin, PHT) were evaluated using 3 week old primary hippocampal cultures derived from 19 day embryonic rat. When added to the culture medium prior to a hypoxic insult, PHT increased neuronal viability two-fold. Doubling extracellular Mg2+ concentration was similarly neuroprotective. In contrast, PHT was unable to protect against hypoxia-induced death in one week old cultures, nor was PHT protective against N-methyl-D-aspartate (NMDA)-induced neurotoxicity in cultures of either age. These findings suggest that non-NMDA receptor mechanisms are important in hypoxia-induced neuronal death, and may have important implications for the treatment of stroke.

Alterations in modulation of acetylcholine release following lesion of hippocampal cholinergic neurons with the neurotoxin AF64A.

Accumulation of acetylcholine (ACh) following administration of physostigmine or tetrahydroaminoacridine (THA) normally inhibits further evoked release of ACh through presynaptic muscarinic receptors. However, in cerebral cortical slices from patients with Alzheimer's disease, ACh release is enhanced by THA, an effect mediated via nicotinic receptors. In this study, the effects of THA and physostigmine were examined in hippocampal slices from rats in which cholinergic neurons were lesioned with the neurotoxin ethylcholine mustard aziridinium (AF64A). Physostigmine and THA did not reduce the evoked release of ACh in lesioned tissues as they did in controls, and THA significantly increased release. The enhancement of release by THA was blocked by the nicotinic antagonist mecamylamine, suggesting that it was mediated through nicotinic receptors. Direct stimulation of muscarinic receptors with oxotremorine significantly reduced ACh release in control tissues, but had no effect in lesioned slices, indicating that presynaptic muscarinic receptors were no longer operative. These results suggest that adaptive changes in nicotinic and muscarinic receptors occur in AF64A-treated rats which are similar to those reported in Alzheimer's disease.

Diphenylhydantoin attenuates hypoxia-induced release of [3H]glutamate from rat hippocampal slices.

The ability of diphenylhydantoin (DPH) to prevent hypoxia-induced [3H]glutamate release was examined in perfused rat hippocampal slices. Hypoxia (25 min; 95% N2/5% CO2) caused a prolonged release of [3H]glutamate, which was reduced significantly if DPH (20 microM) was present from the beginning of the perfusion. Perfusion with oxygenated medium (reoxygenation) following hypoxia also caused a pronounced release of glutamate. A therapeutic concentration of DPH, added before, during, or after hypoxia, decreased this release of glutamate. These results suggest that DPH may protect against glutamate-mediated neurotoxicity associated with stroke.

Dopamine in the rat ventral pallidum/substantia innominata: biochemical and electrophysiological studies.

Recent anatomical literature suggests that dopaminergic projections ascending from the midbrain terminate within the ventral pallidum/substantia innominata (VP/SI). The present investigation evaluated this possibility using standard biochemical and electrophysiologic approaches. Biochemical studies revealed that dopamine and its major metabolites are present within the rat VP/SI. Concentrations of these compounds were diminished greatly when dopaminergic neurons of the substantia nigra were destroyed. Electrophysiologic studies demonstrated that VP/SI neurons often respond to local applications of dopamine with a decrease in firing rate. These observations support the contention that dopamine regulates neuronal activity within the VP/SI and that cells of origin for at least a portion of this projection lie within the substantia nigra.

Inhibition of high affinity choline transport attenuates both cholinergic and non-cholinergic effects of ethylcholine aziridinium (AF64A).

Ethylcholine aziridinium (AF64A) has been proposed as a specific cholinergic neurotoxin. In earlier studies, using AF64A, we reported that slow infusion of 1-2 nmol of this compound into each lateral ventricle of Sprague-Dawley rats resulted in small, and transient decreases in noradrenaline (NA) and serotonin (5-HT) levels in the hippocampus, while inducing a permanent and significant cholinergic hypofunction in the same brain region. The experiments described in this paper were designed to test the hypothesis that such noradrenergic and serotonergic changes after small doses of AF64A are secondary to the changes observed in cholinergic neurons. Levels of NA, and of 5-HT and its metabolite 5-hydroxyindole acetic acid (5-HIAA) were measured concurrently with levels of acetylcholine (ACh), in various brain regions of rats in which the effect of AF64A was attenuated, and in respective control animals. The effect of AF64A was diminished by inhibiting the interaction of AF64A with the high affinity transport site for choline (HAChT). This was achieved using hemicholinium-3 (HC-3), which does not cross the blood-brain barrier, and A-4 (a bis 4-methylpiperidine analog of HC-3), which is centrally active following its peripheral administration. A-4 (20 or 40 mg/kg i.p.) or HC-3 (10 micrograms/ventricle) had no effect on ACh, NA, 5-HT or 5-HIAA levels in saline-treated rats. However, all treatments significantly attenuated the decrease in ACh content produced by AF64A pretreatment. Transient decreases in NA, 5-HT and 5-HIAA contents after AF64A treatment were prevented or reduced by prior treatment with A-4 or HC-3. These results indicate that changes in noradrenergic and serotonergic neurons following AF64A administration are not due to non-specific toxicity of AF64A, but may be the result of adaptation of these neurons to withdrawal of cholinergic input, which would normally inhibit the release of NA and 5-HT. These results also indicate that AF64A can be used to produce specific lesions of hippocampal cholinergic nerve terminals.

Noradrenaline depletion protects cholinergic neurons in rat hippocampus against AF64A-induced damage.

The role of the noradrenergic system in the cholinotoxicity of ethylcholine aziridinium ion (AF64A) was studied in rats. Male Sprague-Dawley rats were treated with the noradrenergic neurotoxin DSP-4 (N-(2-chloroethyl)-n-ethyl-2-bromobenzylamine; 50 mg/kg i.p.) in the presence of the serotonin uptake inhibitor fluoxetine, 14 days prior to bilateral intracerebroventricular injection of AF64A (2 nmol/lateral ventricle). In rats in which noradrenaline (NA) was depleted by 94%, the loss of acetylcholine (ACh) in hippocampus induced by AF64A was significantly attenuated (p less than 0.02). However, when there was only a partial depletion of NA (50% reduction), the AF64A-induced loss of ACh was a pronounced as in rats with intact noradrenergic function. These findings indicate that the noradrenergic lesion has to be complete before a protective effect is apparent. Moreover, they imply that noradrenergic input is involved in AF64A-induced cholinergic damage in the hippocampus.

Clonidine prevents transient loss of noradrenaline in response to cholinergic hypofunction induced by ethylcholine aziridinium (AF64A).

Intracerebroventricular injection of ethylcholine aziridinium (AF64A) (2 nmol/ventricle) induced a considerable decrease in the level of acetylcholine (ACh) in hippocampus (from 21.14 +/- 0.84 to 10.04 +/- 0.59 pmol/mg of tissue; p less than 0.001) 4 days after application. The reduction of cholinergic function was accompanied by a decrease in the level of noradrenaline (NA) (from 1.96 +/- 0.08 to 1.41 +/- 0.06 pmol/mg of tissue; p less than 0.001). Two days after administration of AF64A (1 or 2 nmol/ventricle), the dose-dependent decrease in NA level was associated with an increase in the level of its major metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), resulting in a considerable increase in the MHPG/NA molar ratio (from 0.84 +/- 0.06 to 1.62 +/- 0.17; p less than 0.002). Chronic treatment of AF64A-injected rats with clonidine (0.02-0.2 mg/kg, i.p., every 8-12 h) had no significant effect on the loss of ACh content, whereas the decrease in NA content in hippocampus was completely prevented. Clonidine induced aggressive behavior in the AF64A-treated rats, in contrast to sedation in vehicle-injected rats. The response to clonidine under these experimental conditions and the increased MHPG/NA molar ratio in response to AF64A suggest that the transient loss of NA content following AF64A administration results from increased NA release. The increased noradrenergic activity in hippocampus may be linked to the reduction of tonic inhibitory cholinergic input. These results are discussed in relation to possible implications for senile dementia of the Alzheimer type.

Heterogeneity of presynaptic muscarinic receptors involved in modulation of transmitter release.

In order to extend the characterization of muscarinic receptors at presynaptic sites their inhibitory effect on the stimulation-evoked release of [3H]noradrenaline and [3H]acetylcholine from different axon terminals was studied and the dissociation constants and potencies of different antagonists were estimated, in guinea-pig and rat. While oxotremorine reduced the release of [3H]acetylcholine and [3H]-noradrenaline in a concentration-dependent manner from different release sites (Auerbach plexus, noradrenergic neurons in the right atrium, cerebral cortex), McN-A 343, an M1 receptor agonist, enhanced their release evoked by field stimulation. When the inhibitory effect of oxotremorine on transmitter release was studied, pancuronium, pirenzepine and atropine were competitive antagonists of presynaptic muscarinic receptors located on the noradrenergic axon terminals of the atrium. While atropine and pirenzepine inhibited the muscarinic receptors of cholinergic axon terminals in the Auerbach plexus, pancuronium and gallamine had a very low affinity. Significant differences were found in the affinity constants of antagonists for muscarinic receptors located in the cholinergic axon terminals of Auerbach plexus and cerebral cortex, and noradrenergic axon terminals of the atrium. While atropine and pirenzepine exerted similar effects on these presynaptic sites, pancuronium, gallamine and (11-(2-[diethylamino)-methyl)-1-piperidinyl)acetyl)-5, 11-dihydro-6(1-pyrido(2,3-b)(1,4)-benzodiazepin-6-on) were much more effective on muscarinic receptors controlling acetylcholine release from the cerebral cortex and noradrenaline release from the heart. There was more than 100-fold (2.0 pA2 units) difference in affinities of these antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of LF-14, THA and physostigmine in rat hippocampus and cerebral cortex.

The effects of physostigmine, tetrahydroaminoacridine (THA) and LF-14 [3,3-dimethyl-1(4- amino-3-pyridyl)urea], a 3,4-diaminopyridine derivative, were compared on inhibition of acetyl- cholinesterase (AChE) activity, and release of [(3)H]acetylcholine (ACh) from rat brain cortical and hippocampal slices. All three compounds caused a concentration dependent inhibition of AChE, with an order of potency physostigmine > THA >LF-14. The electrically stimulated release of ACh from hippocampal and cortical slices was decreased by 10(?5)M physostigmine, although the effect was significant only in cortex. THA (5 x 10(5)M) caused a slight, but not significant, decrease in ACh release from both tissues. In contrast, LF-14 (5 x 10(?5) M) caused an approx. 3-fold enhancement of stimulated release. When AChE was inhibited by prior addition of physostigmine, THA caused only a slight enhancement of ACh release, whereas LF-14 greatly increased release. ACh release was also reduced by stimulation of presynaptic muscarinic receptors with oxotremorine. In this case, THA had no effect on ACh release, while LF-14 was able to reverse the inhibition. This study suggests that LF-14 acts to promote ACh release through blocking K(+) channels, and has a less potent AChE inhibitory effect. It is possible that a compound like LF-14 could be useful in treating diseases of cholinergic dysfunction such as Alzheimer's disease, by both promoting the release of ACh and inhibiting its breakdown.