Striatal Acetylcholine Levels Research Articles

Synaptic determinants of cholinergic interneurons hyperactivity during parkinsonism

Parkinson’s disease is a neurodegenerative ailment generated by the loss of dopamine in the basal ganglia, mainly in the striatum. The disease courses with increased striatal levels of acetylcholine, disrupting the balance among these modulatory transmitters. These modifications disturb the excitatory and inhibitory balance in the striatal circuitry, as reflected in the activity of projection striatal neurons. In addition, changes in the firing pattern of striatal tonically active interneurons during the disease, including cholinergic interneurons (CINs), are being searched. Dopamine-depleted striatal circuits exhibit pathological hyperactivity as compared to controls. One aim of this study was to show how striatal CINs contribute to this hyperactivity. A second aim was to show the contribution of extrinsic synaptic inputs to striatal CINs hyperactivity. Electrophysiological and calcium imaging recordings in Cre-mice allowed us to evaluate the activity of dozens of identified CINs with single-cell resolution in ex vivo brain slices. CINs show hyperactivity with bursts and silences in the dopamine-depleted striatum. We confirmed that the intrinsic differences between the activity of control and dopamine-depleted CINs are one source of their hyperactivity. We also show that a great part of this hyperactivity and firing pattern change is a product of extrinsic synaptic inputs, targeting CINs. Both glutamatergic and GABAergic inputs are essential to sustain hyperactivity. In addition, cholinergic transmission through nicotinic receptors also participates, suggesting that the joint activity of CINs drives the phenomenon; since striatal CINs express nicotinic receptors, not expressed in striatal projection neurons. Therefore, CINs hyperactivity is the result of changes in intrinsic properties and excitatory and inhibitory inputs, in addition to the modification of local circuitry due to cholinergic nicotinic transmission. We conclude that CINs are the main drivers of the pathological hyperactivity present in the striatum that is depleted of dopamine, and this is, in part, a result of extrinsic synaptic inputs. These results show that CINs may be a main therapeutic target to treat Parkinson’s disease by intervening in their synaptic inputs.

Prefrontal Cortex-Driven Dopamine Signals in the Striatum Show Unique Spatial and Pharmacological Properties.

Dopamine (DA) signals in the striatum are critical for a variety of vital processes, including motivation, motor learning, and reinforcement learning. Striatal DA signals can be evoked by direct activation of inputs from midbrain DA neurons (DANs) as well as cortical and thalamic inputs to the striatum. In this study, we show that in vivo optogenetic stimulation of prelimbic (PrL) and infralimbic (IL) cortical afferents to the striatum triggers an increase in extracellular DA concentration, which coincides with elevation of striatal acetylcholine (ACh) levels. This increase is blocked by a nicotinic ACh receptor (nAChR) antagonist. Using single or dual optogenetic stimulation in brain slices from male and female mice, we compared the properties of these PrL/IL-evoked DA signals with those evoked by stimulation from midbrain DAN axonal projections. PrL/IL-evoked DA signals are undistinguishable from DAN evoked DA signals in their amplitudes and electrochemical properties. However, PrL/IL-evoked DA signals are spatially restricted and preferentially recorded in the dorsomedial striatum. PrL/IL-evoked DA signals also differ in their pharmacological properties, requiring activation of glutamate and nicotinic ACh receptors. Thus, both in vivo and in vitro results indicate that cortical evoked DA signals rely on recruitment of cholinergic interneurons, which renders DA signals less able to summate during trains of stimulation and more sensitive to both cholinergic drugs and temperature. In conclusion, cortical and midbrain inputs to the striatum evoke DA signals with unique spatial and pharmacological properties that likely shape their functional roles and behavioral relevance.SIGNIFICANCE STATEMENT Dopamine signals in the striatum play a critical role in basal ganglia function, such as reinforcement and motor learning. Different afferents to the striatum can trigger dopamine signals, but their release properties are not well understood. Further, these input-specific dopamine signals have only been studied in separate animals. Here we show that optogenetic stimulation of cortical glutamatergic afferents to the striatum triggers dopamine signals both in vivo and in vitro These afferents engage cholinergic interneurons, which drive dopamine release from dopamine neuron axons by activation of nicotinic acetylcholine receptors. We also show that cortically evoked dopamine signals have other unique properties, including spatial restriction and sensitivity to temperature changes than dopamine signals evoked by stimulation of midbrain dopamine neuron axons.

Striatal and nigral muscarinic type 1 and type 4 receptors modulate levodopa-induced dyskinesia and striato-nigral pathway activation in 6-hydroxydopamine hemilesioned rats

Acetylcholine muscarinic receptors (mAChRs) contribute to both the facilitation and inhibition of levodopa-induced dyskinesia operated by striatal cholinergic interneurons, although the receptor subtypes involved remain elusive. Cholinergic afferents from the midbrain also innervate the substantia nigra reticulata, although the role of nigral mAChRs in levodopa-induced dyskinesia is unknown. Here, we investigate whether striatal and nigral M1 and/or M4 mAChRs modulate dyskinesia and the underlying striato-nigral GABAergic pathway activation in 6-hydroxydopamine hemilesioned rats. Reverse microdialysis allowed to deliver the mAChR antagonists telenzepine (M1 subtype preferring), PD-102807 and tropicamide (M4 subtype preferring), as well as the selective M4 mAChR positive allosteric modulator VU0152100 in striatum or substantia nigra, while levodopa was administered systemically. Dyskinetic movements were monitored along with nigral GABA (and glutamate) and striatal glutamate dialysate levels, taken as neurochemical correlates of striato-nigral pathway and cortico-basal ganglia-thalamo-cortical loop activation. We observed that intrastriatal telenzepine, PD-102807 and tropicamide alleviated dyskinesia and inhibited nigral GABA and striatal glutamate release. This was partially replicated by intrastriatal VU0152100. The M2 subtype preferring antagonist AFDX-116, used to elevate striatal acetylcholine levels, blocked the behavioral and neurochemical effects of PD-102807. Intranigral VU0152100 prevented levodopa-induced dyskinesia and its neurochemical correlates whereas PD-102807 was ineffective. These results suggest that striatal, likely postsynaptic, M1 mAChRs facilitate dyskinesia and striato-nigral pathway activation in vivo. Conversely, striatal M4 mAChRs can both facilitate and inhibit dyskinesia, possibly depending on their localization. Potentiation of striatal and nigral M4 mAChR transmission leads to powerful multilevel inhibition of striato-nigral pathway and attenuation of dyskinesia.

Increased Cholinergic Response in α-Synuclein Transgenic Mice (h-α-synL62).

Pathological accumulation of misfolded α-synuclein (α-syn) in the brain plays a key role in the pathogenesis of Parkinson's disease, leading to neuronal dysfunction and motor disorders. The underlying mechanisms linking α-syn aggregations with neurotransmitter disturbance in Parkinson's brains are not well characterized. In the present study, we investigated transgenic mice expressing an aggregation-prone form of full-length human α-syn (h-α-synL62) linked to a signal sequence. These mice display dopamine depletion and progressive motor deficits. We detected accumulation of α-syn in cholinergic interneurons where they are colocalized with choline acetyltransferase. Using microdialysis, we measured acetylcholine levels in the striatum at baseline and during stimulation in the open field and with scopolamine. While no difference between wild-type and transgenic mice was detected in 3 month old mice, striatal acetylcholine levels at 9 months of age were significantly higher in transgenic mice. Concomitantly, high-affinity choline uptake was also increased while choline acetyltransferase and acetylcholine esterase activities were unchanged. The results suggest a disinhibition of acetylcholine release in α-syn transgenic mice.

Dysfunctional Presynaptic M2 Receptors in the Presence of Chronically High Acetylcholine Levels: Data from the PRiMA Knockout Mouse.

The muscarinic M2 receptor (M2R) acts as a negative feedback regulator in central cholinergic systems. Activation of the M2 receptor limits acetylcholine (ACh) release, especially when ACh levels are increased because acetylcholinesterase (AChE) activity is acutely inhibited. Chronically high ACh levels in the extracellular space, however, were reported to down-regulate M2R to various degrees. In the present study, we used the PRiMA knockout mouse which develops severely reduced AChE activity postnatally to investigate ACh release, and we used microdialysis to investigate whether the function of M2R to reduce ACh release in vivo was impaired in adult PRiMA knockout mice. We first show that striatal and hippocampal ACh levels, while strongly increased, still respond to AChE inhibitors. Infusion or injection of oxotremorine, a muscarinic M2 agonist, reduced ACh levels in wild-type mice but did not significantly affect ACh levels in PRiMA knockout mice or in wild-type mice in which ACh levels were artificially increased by infusion of neostigmine. Scopolamine, a muscarinic antagonist, increased ACh levels in wild-type mice receiving neostigmine, but not in wild-type mice or in PRiMA knockout mice. These results demonstrate that M2R are dysfunctional and do not affect ACh levels in PRiMA knockout mice, likely because of down-regulation and/or loss of receptor-effector coupling. Remarkably, this loss of function does not affect cognitive functions in PRiMA knockout mice. Our results are discussed in the context of AChE inhibitor therapy as used in dementia.

Changes in extracellular striatal acetylcholine and brain seizure activity following acute exposure to nerve agents in freely moving guinea pigs

Organophosphorus nerve agents irreversibly inhibit acetylcholinesterase (AChE) in the peripheral and central nervous systems, causing an increase in the concentration of acetylcholine (ACh) in the synapse or neuromuscular junction and subsequent adverse effects. In this study, in vivo microdialysis was utilized to collect samples from the striatum for monitoring changes in extracellular ACh levels along with cortical electroencephalographic (EEG) recordings for identifying seizure activity after acute subcutaneous (s.c.) exposure to 1.0 × LD50 of the nerve agents sarin, soman, or one of two V-type agents (VX, or a Russian V-agent, designated VR) in unanesthetized freely moving guinea pigs. Based on EEG recordings, these animals were subsequently divided into groups that developed seizures (S) and those that did not develop seizures (NS). Maximum ACh levels in the striatum were observed at 60–70 min for sarin and soman S groups and 105 min for VX and VR S groups. In all NS groups the greatest increase in extracellular ACh occurred within 30 min after exposure, although in the sarin NS group a few sporadic increases of ACh from control occurred. Animals that developed seizures, regardless of the nerve agent, had significantly higher extracellular striatal ACh levels compared to the controls or those animals that did not develop seizures, yet both S and NS groups displayed similar levels of blood AChE inhibition. Regardless of the agent, all animals in the non-seizure groups survived 24 h, while lethality (25–42%) was observed only in animals that experienced seizure activity.

Impairment of bidirectional synaptic plasticity in the striatum of a mouse model of DYT1 dystonia: role of endogenous acetylcholine

DYT1 dystonia is a severe form of inherited dystonia, characterized by involuntary twisting movements and abnormal postures. It is linked to a deletion in the dyt1 gene, resulting in a mutated form of the protein torsinA. The penetrance for dystonia is incomplete, but both clinically affected and non-manifesting carriers of the DYT1 mutation exhibit impaired motor learning and evidence of altered motor plasticity. Here, we characterized striatal glutamatergic synaptic plasticity in transgenic mice expressing either the normal human torsinA or its mutant form, in comparison to non-transgenic (NT) control mice. Medium spiny neurons recorded from both NT and normal human torsinA mice exhibited normal long-term depression (LTD), whereas in mutant human torsinA littermates LTD could not be elicited. In addition, although long-term potentiation (LTP) could be induced in all the mice, it was greater in magnitude in mutant human torsinA mice. Low-frequency stimulation (LFS) can revert potentiated synapses to resting levels, a phenomenon termed synaptic depotentiation. LFS induced synaptic depotentiation (SD) both in NT and normal human torsinA mice, but not in mutant human torsinA mice. Since anti-cholinergic drugs are an effective medical therapeutic option for the treatment of human dystonia, we reasoned that an excess in endogenous acetylcholine could underlie the synaptic plasticity impairment. Indeed, both LTD and SD were rescued in mutant human torsinA mice either by lowering endogenous acetylcholine levels or by antagonizing muscarinic M1 receptors. The presence of an enhanced acetylcholine tone was confirmed by the observation that acetylcholinesterase activity was significantly increased in the striatum of mutant human torsinA mice, as compared with both normal human torsinA and NT littermates. Moreover, we found similar alterations of synaptic plasticity in muscarinic M2/M4 receptor knockout mice, in which an increased striatal acetylcholine level has been documented. The loss of LTD and SD on one hand, and the increase in LTP on the other, demonstrate that a 'loss of inhibition' characterizes the impairment of synaptic plasticity in this model of DYT1 dystonia. More importantly, our results indicate that an unbalanced cholinergic transmission plays a pivotal role in these alterations, providing a clue to understand the ability of anticholinergic agents to restore motor deficits in dystonia.

Effects of stimulation of the centromedian nucleus of the thalamus on the activity of striatal cells in awake rhesus monkeys

Although the existence of a massive projection from the caudal intralaminar nuclei of the thalamus [i.e. the centromedian (CM) and parafascicular nuclei] to the striatum is well documented, the effects of CM activation upon striatal cells remain poorly understood. Therefore, we studied the effects of electrical stimulation of CM on the electrophysiological activity of striatal neurons, and on striatal levels of gamma-aminobutyric acid (GABA) and acetylcholine in rhesus monkeys. Striatal cells did not respond to single-pulse stimulation (bipolar biphasic stimulation, 175-500 muA), but the large majority of recorded neurons responded to burst stimulation (100 Hz, 1 s, 150-175 muA) of CM, often with a delay of tens of milliseconds. Striatal phasically active neurons, which likely correspond to projection neurons, responded mainly with increases in firing (13/28 cells), while tonically active neurons (likely cholinergic interneurons) often showed combinations of increases and decreases in firing (24/46 cells). In microdialysis studies, CM stimulation led to a reduction of striatal acetylcholine levels. This effect was prevented by addition of the GABA-A receptor antagonist gabazine to the microdialysis fluid. We conclude that CM stimulation frequently results in striatal response patterns with excitatory and inhibitory components. Under the conditions chosen here, the specific patterns of striatal responses to CM stimulation are likely the result of striatal processing of thalamic inputs. Through these indirect effects, local CM stimulation may engage large portions of the striatum. These effects may be relevant in the interpretation of the therapeutic effects of CM stimulation for the treatment of neurological disorders.

Comparative in vivo effects of parathion on striatal acetylcholine accumulation in adult and aged rats

Aged rats are more sensitive to the acute toxicity of the prototype organophosphate insecticide, parathion. We compared the acute effects of parathion on diaphragm and brain regional cholinesterase activity, muscarinic receptor binding and striatal acetylcholine levels in 3- and 18-month-old male Sprague–Dawley rats. Adult and aged rats were surgically implanted with a microdialysis cannula into the right striatum 5–7 days prior to parathion treatment. Rats were given either vehicle (peanut oil, 2 ml/kg) or one of a range of dosages of parathion (adult: 1.8, 3.4, 6.0, 9.0, 18 and 27 mg/kg, s.c.; aged: 1.8, 3.4, 6 and 9 mg/kg, s.c.) and body weight, functional signs of toxicity, and nocturnal motor activity were recorded for seven days. Three and seven days after parathion treatment, microdialysis samples were collected and rats were subsequently sacrificed for biochemical measurements. Higher dosages of parathion led to significant time-dependent reductions in body weight in both age groups. Rats in both age groups treated with lower dosages showed few overt signs of cholinergic toxicity while equitoxic high dosages (adult, 27 mg/kg; aged, 9 mg/kg) elicited marked signs of cholinergic toxicity (involuntary movements and SLUD [i.e., acronym for Salivation, Lacrimation, Urination and Defecation] signs) with peak effects being noted 3–4 days after treatment. Nocturnal activity (ambulation and rearing) was reduced in both age groups following parathion dosing, with more prominent effects in adults and rearing being more consistently affected. Dose- and time-dependent inhibition of cholinesterase activity was noted in both diaphragm and striatum. Total muscarinic receptor ([ 3H]quinuclidinyl benzilate, QNB) binding was significantly lower in aged rats, and both total binding and muscarinic agonist ([ 3H]oxotremorine methiodide] binding was significantly reduced in both age-groups treated with the highest dosages of parathion (adult, 27 mg/kg; aged, 9 mg/kg). In contrast to relatively similar levels of cholinesterase inhibition, striatal extracellular acetylcholine levels were significantly lower (2.2- to 2.9-fold) in aged rats at both 3 and 7 day time-points compared to adult rats treated with equitoxic dosages (i.e., 9 and 27 mg/kg, respectively). No age-related differences in in vitro striatal acetylcholine synthesis or in vivo acetylcholine accumulation following direct infusion of the cholinesterase inhibitor neostigmine (1 μM) were noted. While aged rats are more sensitive than adults to the acute toxicity of parathion, lesser acetylcholine accumulation was noted in the striatum of aged rats exhibiting similar levels of cholinesterase inhibition. These findings suggest that lesser acetylcholine accumulation may be required to elicit cholinergic signs in the aged rat, possibly based on aging-associated changes in muscarinic receptor density.

Effects of acute chlorpyrifos exposure on in vivo acetylcholine accumulation in rat striatum

This study examined the acute effects of chlorpyrifos (CPF) on cholinesterase inhibition and acetylcholine levels in the striatum of freely moving rats using in vivo microdialysis. Adult, male Sprague-Dawley rats were treated with vehicle (peanut oil, 2 ml/kg) or CPF (84, 156 or 279 mg/kg, sc) and functional signs of toxicity, body weight and motor activity recorded. Microdialysis was conducted at 1, 4 and 7 days after CPF exposure for measurement of acetylcholine levels in striatum. Rats were then sacrificed and the contralateral striatum and diaphragm were collected for biochemical measurements. Few overt signs of cholinergic toxicity were noted in any rats. Body weight gain was significantly affected in the high-dose (279 mg/kg) group only, while motor activity (nocturnal rearing) was significantly reduced in all CPF-treated groups at one day (84 mg/kg) or from 1-4 days (156 and 279 mg/kg) after dosing. Cholinesterase activities in both diaphragm and striatum were markedly inhibited (50-92%) in a time-dependent manner, but there were relatively minimal dose-related changes. In contrast, time- and dose-dependent changes in striatal acetylcholine levels were noted, with significantly higher levels noted in the high-dose group compared to other groups. Maximal increases in striatal acetylcholine levels were observed at 4-7 days after dosing (84 mg/kg, 7-9-fold; 156 mg/kg, 10-13-fold; 279 mg/kg, 35-57-fold). Substantially higher acetylcholine levels were noted when an exogenous cholinesterase inhibitor was included in the perfusion buffer, but CPF treatment-related differences were substantially lower in magnitude under those conditions. The results suggest that marked differences in acetylcholine accumulation can occur with dosages of CPF eliciting relatively similar degrees of cholinesterase inhibition. Furthermore, the minimal expression of classic signs of cholinergic toxicity in the presence of extensive brain acetylcholine accumulation suggests that some compensatory process(es) downstream from synaptic neurotransmitter accumulation limits the expression of toxicity following acute CPF exposure.

The effects of repeated low-dose sarin exposure☆

This project assessed the effects of repeated low-dose exposure of guinea pigs to the organophosphorus nerve agent sarin. Animals were injected once a day, 5 days per week (Monday-Friday), for 2 weeks with fractions (0.3x, 0.4x, 0.5x, or 0.6x) of the established LD(50) dose of sarin (42 microg/kg, s.c.). The animals were assessed for changes in body weight, red blood cell (RBC) acetylcholinesterase (AChE) levels, neurobehavioral reactions to a functional observational battery (FOB), cortical electroencephalographic (EEG) power spectrum, and intrinsic acetylcholine (ACh) neurotransmitter (NT) regulation over the 2 weeks of sarin exposure and for up to 12 days postinjection. No guinea pig receiving 0.3, 0.4 or 0.5 x LD(50) of sarin showed signs of cortical EEG seizures despite decreases in RBC AChE levels to as low as 10% of baseline, while seizures were evident in animals receiving 0.6 x LD(50) of sarin as early as the second day; subsequent injections led to incapacitation and death. Animals receiving 0.5 x LD(50) sarin showed obvious signs of cholinergic toxicity; overall, 2 of 13 animals receiving 0.5 x LD(50) sarin died before all 10 injections were given, and there was a significant increase in the angle of gait in the animals that lived. By the 10th day of injection, the animals receiving saline were significantly easier to remove from their cages and handle and significantly less responsive to an approaching pencil and touch on the rump in comparison with the first day of testing. In contrast, the animals receiving 0.4 x LD(50) sarin failed to show any significant reductions in their responses to an approaching pencil and a touch on the rump as compared with the first day. The 0.5 x LD(50) sarin animals also failed to show any significant changes to the approach and touch responses and did not adjust to handling or removal from the cage from the first day of injections to the last day of handling. Thus, the guinea pigs receiving the 0.4 and 0.5 x LD(50) doses of sarin failed to habituate to some aspects of neurobehavioral testing. Spectral analysis of EEG data suggested that repeated sarin exposure may disrupt normal sleeping patterns (i.e., lower frequency bandwidths). While these EEG changes returned to relative normalcy 6 days after the last injection in animals receiving 0.4 x LD(50) sarin, these changes were still observed in the animals that received 0.5 x LD(50) sarin. Ten to twelve days after the last sarin injection (in 0.4 x LD(50) group only), neurochemical data showed that striatal choline levels were reduced in comparison to the saline group. At this time, atropine sulfate (5 mg/kg, i.p.) challenge resulted in a transient elevation in striatal ACh levels in animals exposed to repeated 0.4 x LD(50) sarin as well as in control animals. No evidence of brain or heart pathology was found in any guinea pig that survived all 10 sarin injections.

Sumanirole, a Highly Dopamine D2-Selective Receptor Agonist: In Vitro and in Vivo Pharmacological Characterization and Efficacy in Animal Models of Parkinson's Disease

The purpose of this study is to demonstrate that sumanirole is a novel dopamine receptor agonist with high in vitro and in vivo selectivity for the D(2) receptor subtype. Sumanirole, (R)-5,6-dihydro-5-(methylamino)-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (Z)-2-butenedioate (1:1), is unique; it has greater than 200-fold selectivity for the D(2) receptor subtype versus the other dopamine receptor subtypes in radioligand binding assays. In cell-based assays, sumanirole is a fully efficacious agonist, with EC(50) values between 17 and 75 nM. In animals, sumanirole elicits many physiological responses attributed to D(2)-like receptor function. In rats, sumanirole is a full agonist for elevation of striatal acetylcholine levels (ED(50) = 12.1 micromol/kg i.p.). Sumanirole s.c. dose dependently decreased plasma prolactin levels and depressed dopamine neuron firing rates in the substantia nigra pars compacta with an ED(50) of 2.3 micromol/kg i.v. This high selectivity for D(2) receptors translates into excellent locomotor stimulant activity in animal models of Parkinson's disease. In reserpinized, alpha-methyl-para-tyrosine-treated rats, sumanirole caused a significant and sustained increase in horizontal activity at doses > or =12.5 micromol/kg s.c. In unilateral 6-hydroxydopamine-lesioned rats, sumanirole caused profound, sustained rotational behavior and was substantially more efficacious than any other agonist tested. Sumanirole-stimulated rotational behavior was blocked by the dopamine receptor antagonist haloperidol. Sumanirole dose dependently improved disability scores and locomotor activities of two of three 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned monkeys. In summary, sumanirole is the first published selective D(2) receptor agonist. The compound has activity in animal models of dopamine hypofunction and has a high level of efficacy in animal models of Parkinson's disease.

Effects of carbamazepine on acetylcholine release and metabolism

To clarify the mechanisms of action of carbamazepine (CBZ), we investigated the effects of CBZ on acetylcholine (ACh) release and metabolism in rat striatum and hippocampus. Acute administration of effective dose of CBZ (25 mg/kg) increased both striatal and hippocampal extracellular levels of ACh, whereas a supraeffective dose of CBZ (50 mg/kg) did not affect the levels and a toxic dose of CBZ (100 mg/kg) decreased the extracellular ACh levels in both brain regions. Both acute and chronic administrations of CBZ (25 and 50 mg/kg, mg/kg per day) increased intracellular ACh levels in striatum and hippocampus. The striatal intracellular ACh levels were decreased by both acute and chronic administrations of CBZ (100 mg/kg, mg/kg per day), whereas the hippocampal intracellular ACh levels were not affected. The effective CBZ concentration did not affect cholinesterase activity, whereas supraeffective CBZ concentration reduced it weakly. Effective dose of CBZ enhanced ACh release and synthesis; however, supraeffective doses of CBZ reduced ACh release and synthesis without enhancement of ACh degradation, indicating that CBZ has biphasic effects on ACh release and synthesis. Thus, the present findings, the slight stimulation of ACh function by effective dose of CBZ, are involved, at least partially, in the antiepileptic and mood stabilizing mechanisms of action of CBZ.

Pharmacological role of isatin, an endogenous MAO inhibitor

Isatin (indole-2,3-dione) has been found in mammalian tissues as one of major components of tribulin, a postulated endogenous marker of stress and anxiety. I previously identified isatin as an endogenous inhibitor of monoamine oxidase (MAO) in the human urine and the brain of stroke-prone spontaneously hypertensive rats (SHRSP) using GC-MS. A single dose of isatin significantly increased norepinephrine (NE) and 5-hydroxytryptamine (5-HT) concentrations measured 2 h later in the various brain regions of normotensive Wistar Kyoto rats (WKY). Striatal acetylcholine (ACh) and dopamine (DA) levels significantly increased 2 h after the administration of isatin. Perfused through a microdialysis probe, isatin also produced a significant and concentration-dependent increase in the ACh and DA concentration in the perfusate from the rat striatum. In the patients with Parkinson's disease, urinary isatin concentrations tended to increase according to the severity of disease, as classified by the Hoehn and Yahr criteria. Isatin significantly increased striatal DA levels in a rat model of Parkinson's disease. Isatin may play a role in the regulation of the brain levels of ACh and DA. Furthermore, isatin has a wide spectrum of biological properties: (a) a marker of stress and anxiety, (b) an inhibitor of a number of enzymes, (c) an anti-seizure agent, (d) an inhibitor of benzodiazepin receptors and ANP binding to its receptors.

The Role of Ventrolateral Striatal Acetylcholine in the Production of Tacrine-Induced Jaw Movements

The anticholinesterase tacrine induces tremulous jaw movements in rats, and considerable evidence indicates that this response is dependent upon ventrolateral striatal mechanisms. Three experiments were conducted to study the relation between ventrolateral striatal acetylcholine and the production of tremulous jaw movements. In Experiment 1, intracranial microinjection of the acetylcholine synthesis inhibitor hemicholinium-3 into the ventrolateral neostriatum reduced tremulous jaw movements induced by 5.0 mg/kg tacrine. Microinjection of hemicholinium into a cortical site dorsal to striatum (Experiment 2) was without significant effect upon tacrine-induced tremulous jaw movements. In Experiment 3, rats were implanted with dialysis probes in the ventrolateral striatum to measure extracellular levels of acetylcholine during tacrine-induced jaw movements. Tacrine (2.5–5.0 mg/kg) increased both extracellular acetylcholine and tremulous jaw movements. The 5.0 mg/kg dose of tacrine produced a substantial increase in ventrolateral striatal acetylcholine levels (324% of baseline within 30 min). Across all tacrine-treated rats there was a significant linear correlation between tremulous jaw movements and acetylcholine levels ( r = +0.56) during the first 30-min postinjection period. This correlation was largely due to the group that received 5.0 mg/kg tacrine; within this group, there was a very high correlation ( r = +0.87) between tremulous jaw movements and acetylcholine levels in the first sample after injection. These data are consistent with the notion that tremulous jaw movements induced by tacrine are mediated by ventrolateral striatal acetylcholine. Moreover, these results suggest that dialysis methods could be used to monitor the relation between striatal acetylcholine and tremulous movements induced by a variety of different conditions.

Effects of isatin, an endogenous MAO inhibitor, on dopamine (DA) and acetylcholine (ACh) concentrations in rats

Isatin (indole-2,3-dione), an endogenous inhibitor of monoamine oxidase (MAO), has several physiological properties for stress and anxiety. We previously identified isatin in the brain of stroke-prone spontaneously hypertensive rats (SHRSP) using gas-chromatography mass spectrometry. This study elucidated the effects of isatin on the ACh and DA levels of brain tissues in rats. Furthermore, we evaluated the effect of isatin on DA levels in a rat model of Parkinson's disease induced by Japanese encephalitis virus. Striatal ACh and DA levels significantly increased at 2 hours after isatin (50-200 mg/kg, i.p.) administration. Perfused through a microdialysis probe, isatin (10(-6)-10(-4) M) also produced a significant and concentration-dependent increase in the ACh and DA concentrations in the perfusate from the rat striatum. Furthermore, urinary isatin concentrations in patients with Parkinson's disease tend to increase according to the severity of disease. Isatin (100 mg/kg, i.p.) significantly increased striatal DA levels in a rat model of Parkinson's disease. These results suggest that urinary isatin may become a diagnostic marker for the clinical severity of Parkinson's disease and that endogenous isatin, a new biological modulator, may play a role in the regulation of the brain levels of ACh by increasing the level of DA under stress.

Differential effects of the neuropeptide galanin on striatal acetylcholine release in anaesthetized and awake rats.

1. In the present study the mechanisms were examined by which the neuropeptide galanin modulates the extracellular concentrations of striatal acetylcholine (ACh) in enflurane anaesthetized and in freely moving male rats by use of in vivo microdialysis and high performance liquid chromatography. 2. The perfusion of galanin through the microdialysis probe (0.3 nmol microl(-1), flow rate: 2 microl min(-1)) caused a statistically significant increase in the basal striatal ACh levels in anaesthetized but a decrease in awake animals. No significant effect was revealed after a low dose (0.1 nmol microl(-1), flow rate: 2 microl min(-1)) of galanin perfusion. Both the stimulating and inhibitory effects of galanin on basal ACh release were reversible. 3. The muscarinic antagonist scopolamine (0.1 mg kg(-1), subcutaneously (s.c.)) caused a significant increase in ACh release in both anaesthetized and awake animals. 4. The combination of galanin plus scopolamine attenuated the stimulant effect on ACh release caused by scopolamine alone in awake animals. 5. The putative galanin receptor antagonist M35 at 0.3 nmol microl(-1) but not at 0.1 nmol microl(-1) caused a significant reduction (20%) in ACh release, supporting the view that M35 at higher concentrations behaves as a partial agonist at the galanin receptor. When M35 (0.1 nmol microl(-1)) was co-infused with galanin (0.3 nmol microl(-1)) the galanin-evoked decrease in ACh release was completely blocked. 6. Taken together, these results indicate that galanin affects basal ACh release via stimulation of galanin receptors within the striatum. The mechanism involved is dependent on the anaesthesia procedure which may act via enhancement of gamma-aminobutyric acidA (GABA(A)) mediated transmission within striatal and/or output neurones. In addition, anaesthesia may also decrease the activity of glutamatergic striatal afferents. The results with M35 indicate that the role of galanin perfused in striatum is permissive in the normal rat. Furthermore, galanin is a potent inhibitory modulator of basal ACh release also in the striatum, as recently was shown in the ventral hippocampus in awake animals.

SA4503, a novel cognitive enhancer, with σ1 receptor agonistic properties

We found a potent and selective sigma1 (σ1) receptor ligand, SA4503 (1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride). This compound had a high affinity for σ1 receptor subtype (IC50=17±1.9 nM), but a low affinity for σ2 receptor subtype (IC50 = 1800 ± 310 nM). The present study examines the effect of this compound on the central cholinergic functions, since σ receptor has been reported to interact with the central cholinergic neurons. SA4503 elicited the increase in extracellular acetylcholine level in rat frontal cortex, while it did not affect the striatal acetylcholine level. On the other hand, tetrahydroaminoacridine (THA), an acetylcholinesterase (AChE) inhibitor, increased the extracellular acetylcholine level in both regions. Although both compounds had anti-amnesic effect against scopolamine-induced memory impairment, THA also induced catalepsy in rats. These results suggest that SA4503 may be a novel cognitive enhancer, with σ1 receptor agonistic properties. In addition, SA4503 does not cause striatal cholinomimetic side-effects, which is different from THA.

D 2 and D 1 dopaminergic activity of 7-OH-DPAT

The D 1 and D 2 dopamine receptor agonist properties of 7-hydroxy-2-( N,N-di- n-propylamino) tetraline (7-OH-DPAT) was determined by investigating the effect of this compound on rat striatal acetylcholine (ACh) concentration and increase in cAMP formation in primary cerebellar granule cell cultures. 7-OH-DPAT at low doses (0.01 to 0.1 μmol/kg) had no significant effect, and at high doses (0.3 to 30 μmol/kg) significantly ( P < 0.01) increased striatal ACh levels. Likewise, quinpirole was found to significantly elevate ACh content. Pretreatment with haloperidol, a non-selective antagonist of the D2 family of receptors, significantly ( P < 0.01) blocked 7-OH-DPAT- and quinpirole-induced increases in ACh. U-99194A, a D 3 selective dopamine antagonist, had no significant effect on 7-OH-DPAT-induced increases in striatal ACh. However, raclopride, a D 2 selective dopamine antagonist, completely blocked 7-OH-DPAT-induced elevations in ACh. 7-OH-DPAT in the μmolar range increased cAMP formation in granule cell cultures, and this effect was antagonized by SCH 23390, a D 1 selective dopamine antagonist. The neurochemical study indicates that, at high doses, 7-OH-DPAT has both D 1 and D 2 agonist activities.

Regional differences of the effect of sigma receptor ligands on the acetylcholine release in the rat brain.

We found that sigma receptor ligands differentially regulated the acetylcholine (ACh) neurotransmission in the rat brain. Acute administration of (+)-N-allylnormetazocine [(+)-SKF-10,047], a prototype sigma 1 receptor ligand, and 1,3-di(2-tolyl)guanidine (DTG), a non-specific sigma receptor ligand, increased the extracellular ACh level in the rat hippocampus. This increase of hippocampal extracellular ACh level elicited by (+)-SKF-10,047 was more potent than that elicited by DTG. On the other hand, the striatal extracellular ACh level was slightly affected by (+)-SKF-10,047. In addition, DTG did not affect the striatal extracellular ACh level. Our previous studies have shown that both (+)-SKF-10,047 and DTG increased the extracellular ACh level in the rat frontal cortex. Taking all these data into consideration, the regulation of ACh neurotransmission by sigma receptor ligands are different depending upon the brain region.