Forskolin attenuates the evoked release of [H]GABA from striatal neurons in primary culture

The actions of the diterpene forskolin, and cyclic AMP analogues, on the evoked release of [³H]GABA (γ-aminobutyric acid) was examined in intact striatal neurons in primary culture, generated from the fetal mouse brain. Exposure of striatal neurons to forskolin (100 μM) resulted in a 40–55% attenuation of [³H]GABA release evoked by either KCl (30 mM) or veratrine (2 μg/ml), while baseline levels of release were unaffected. The dose-dependence for forskolin in striatal neurons. Exposure of striatal neurons to membrane-permeable identical to the dose-dependent elevation of cyclic AMP levels by forskolin in striatal neurons. Exposure of striatal neurons to membrane-permeable analogues of cyclic AMP, such as p-chlorophenylthio cyclic AMP (0.5 mM) and dibutyryl cyclic AMP (1 mM), resulted in a 25 and 26% attenuation of [³H]GABA release, respectively; dibutyryl cyclic GMP (1 mM) was without effect. The similarity between the actions of forskolin and the cyclic AMP analogues suggests that, in striatal neurons in primary culture, the elevation of cyclic AMP levels results in the attenuation of the evoked release of [³H]GABA. The greater effectiveness of forskolin, compared to the cyclic AMP analogues, may be related to the recently reported, additional direct actions of forskolin on neuronal membrane ion channels.     

Biphasic effect of tacrine on acetylcholine release in rat brain via M1 and M2 receptors

Rat cortical synaptosomes preloaded with [³H]choline were superfused and stimulated with K⁺ in order to investigate the effect of the cholinesterase inhibitor tacrine on the in vitro release of acetylcholine (ACh). Tacrine was found to biphasically both increase (10⁻⁶ and 5 × 10⁻⁶ M) and decrease (10⁻⁵−10-⁻⁴ M) the release of ACh in a concentration-dependent manner. The facilitatory effect of tacrine was prevented by atropine and the M₁ antagonist pirenzepine, whereas the inhibitory effect induced by tacrine was blocked by atropine and the M₂ antagonist AF-DX 116. These results indicate that tacrine causes a biphasic effect on K⁺ stimulated ACh release in the brain via M₁ and M₂ muscarinic receptors. The tacrine induced enhancement of the ACh release occurs at clinical relevant tacrine concentrations and might therefore be of importance for the treatment outcome of Alzheimer's disease.     

Competitive inhibition of phosphoinositide hydrolysis by the muscarinic receptor antagonist AF-DX 116 is of low affinity in mouse cerebral cortex

Carbamylcholine stimulated [³H]inositol phosphate accumulation in mouse cerebral cortical slices with an ED₅₀ value of approximately 70 μM. Increasing concentrations of the M₂ selective muscarinic cholinergic receptor antagonist, AF-DX 116 (0.3–3.0 μM), produced parallel shifts to the right for concentration-response curves to carbamylcholine. A pA₂ value for AF-DX 116 of 6.5 (low affinity) was obtained frommSchild plot analysis. It is concluded that the M₂ muscarinic receptor subtype, as defined by high affinity [³H]AF-DX 116 radioligand binding, is not appreciably coupled to polyphosphoinositide hydrolysis in the mouse cerebral cortex.     

α2-Adrenergic receptors mediate inhibition of cyclic AMP production in neurons in primary culture

The actions of adrenergic agents on the intracellular production of cyclic adenosine monophosphate (AMP) was examined in intact cortical and striatal neurons in primary culture, generated from the fetal mouse brain. Exposure of striatal neurons to the β-adrenergic agonist isoproterenol (10 μM) resulted in a 5-fold increase in intraneuronal cyclic AMP; norepinephrine (100 μM), alone or in combination with isoproterenol, produced only a 3-fold increase in cyclic AMP levels. However, in the presence of yohimbine (10 μM), cyclic AMP productions due to norepinephrine or isoproterenol plus norepinephrine were identical to isoproterenol alone. When striatal or cortical neurons were exposed to pertussis toxin (100 ng/ml) overnight, there was no detectable difference between isoproterenol- and norepinephrine-stimulated cyclic AMP production. These data suggest thatα₂-adrenergic receptors mediate the attenuation of cyclic AMP production in neurons and do so via the inhibitory guanine nucleotide regulatory protein of adenylate cyclase.     

Cryopreservation of primary neurons for tissue culture

We have developed new cryopreservation methods which allow storage of fetal rat central nervous system tissues for more than 1 week at 3-8 degrees C or for several months at -70 or -90 degrees C prior to tissue culture. For refrigeration, small brain regions (less than 2 mm thick) were placed intact into 35 mm petri dishes of 'hibernation medium' inside a humidified chamber. Optimal preservation was obtained with hibernation media of pH 6.8-7.4, containing 30-70 mM K+, 10-30 mM Na+, 5-50 mM PO4(2-), 20 mM lactic acid, 5 mM glucose, and less than 0.1 mM Ca2+. The media were made approximately isotonic by addition of sorbitol. For freezing, brain tissues were dissociated by gentle trituration (without enzymes) in the above medium supplemented with 5-10% dimethylsulfoxide. After refrigeration or freezing, neurons were very sensitive to damage from mechanical stress (e.g. centrifugation, harsh rinsing or trituration). Rapid changes in osmotic pressure or excessive polylysine on the tissue culture substratum also reduced neuronal survival after cryopreservation. Pretreatment of tissue culture substrata with media from lung cell cultures, or plating of neurons at higher density (2000 cells/mm2) improved neuronal survival after cryopreservation.     

Presynaptic inhibitory receptors mediate the depression of synaptic transmission upon hypoxia in rat hippocampal slices

Hypoxia markedly depresses synaptic transmission in hippocampal slices of the rat. This depression is attributed to presynaptic inhibition of glutamate release and is largely mediated by adenosine released during hypoxia acting through presynaptic adenosine A(1) receptors. Paired pulse facilitation studies allowed us to confirm the presynaptic nature of the depression of synaptic transmission during hypoxia. We tested the hypothesis that activation of heterosynaptic inhibitory receptors localized in glutamatergic presynaptic terminals in the hippocampus, namely gamma-aminobutyric acid subtype B (GABA(B)) receptors, alpha(2)-adrenergic receptors, and muscarinic receptors might contribute to the hypoxia-induced depression of synaptic transmission. Field excitatory postsynaptic potentials were recorded in the CA1 area of hippocampal slices from young adult (5-6 weeks) Wistar rats. Neither the selective antagonist for alpha(2)-adrenergic receptors, rauwolscine (10 microM), nor the antagonist for the GABA(B) receptors, CGP 55845 (10 microM), modified the response to hypoxia. The selective adenosine A(1) receptor antagonist, DPCPX (50 nM), reduced the hypoxia-induced depression of synaptic transmission to 59.2+/-9.6%, and the muscarinic receptor antagonist, atropine (10 microM), in the presence of DPCPX (50 nM), further attenuated the depression of synaptic transmission to 49.4+/-8.0%. In the same experimental conditions, in the presence of DPCPX (50 nM), the muscarinic M(2) receptor antagonist AF-DX 116 (10 microM), but not the M(1) receptor antagonist pirenzepine (1 microM), also attenuated the hypoxia-induced depression to 41.6+/-6.6%. Activation of muscarinic M(2) receptors contributes to the depression of synaptic transmission upon hypoxia. This effect should assume particular relevance during prolonged periods of hypoxia when other mechanisms may become less efficient.     

L-多聚赖氨酸对原代海马神经元生长影响

目的 探讨不同相对分子质量的左旋(L-)多聚赖氨酸对新生鼠原代海马神经元生长的影响. 方法 采用针蕊挤压过网法制备游离新生鼠海马神经细胞悬液,将细胞分为3组,分别种植于未包被、相对分子质量70 000～150 000及相对分子质量150 000～300 000的L-多聚赖氨酸包被的培养板内.Neurobasal-A选择性培养基维持培养,观察不同阶段神经元的形态,神经元特异性烯醇化酶免疫荧光染色鉴定并计算纯度,观察不同相对分子质量的L-多聚赖氨酸对神经元生长特性的影响. 结果 神经元种植1h后开始贴壁;4d后神经元胞体透亮,结构完整,突触长出2～3支;8d后胞体成熟,突触增多,交织成网.神经元特异性烯醇化酶免疫荧光染色鉴定为阳性,神经元平均纯度为92.6％+4.62％.无L-多聚赖氨酸包被的孔内细胞少量存活;相对分子质量70 000～150 000的L-多聚赖氨酸包被的孔内细胞抱团样生长;相对分子质量150 000～300 000的L-多聚赖氨酸包被的孔内细胞生长成均匀的单层,较少有抱团现象. 结论 不同相对分子质量的L-多聚赖氨酸影响神经元的贴附和行为,相对分子质量150 000～300 000的L-多聚赖氨酸最适宜神经元的生长.     

Differential effects of M1 and M2 muscarinic drugs on septohippocampal, hippocampal and cortical neurons in the rat

The effects of muscarinic agonists (McN-A-343, pilocarpine, oxotremorine-M, carbachol) and antagonists (pirenzepine, gallamine) applied by iontophoresis were studied on several neuronal populations in the central nervous system of rats anesthetized with urethane. Septohippocampal neurons and neurons from hippocampus, subiculum and somatic sensory cortex were studied. Oxotremorine-M and carbachol had (almost exclusively) potent excitatory effects whereas pilocarpine had some and McN-A-343 had almost exclusively inhibitory effects on the 4 populations of neurons studied. Pirezepine blocked more easily the effects of pilocarpine and McN-A-343 than those of oxotremorine-M or carbachol. These results suggest (i) that many central neurons may bear different functional muscarinic receptors and (ii) that the various agonists studied might act through (at least partially) different mechanisms.     

Acetylcholine modifies neuronal acoustic rate-level functions in guinea pig auditory cortex by an action at muscarinic receptors

Cholinergic modification of neuronal responsiveness in auditory cortex includes alteration of spontaneous and tone-evoked neuronal discharge. Previously it was suggested that the effects of acetylcholine (ACh) and muscarinic agonists on neuronal discharge resembled those due to increases in the intensity of acoustic stimuli (Ashe et al. 1989). To determine the relationship between neuronal modifications due to ACh acting at muscarinic receptors and those due to changes in stimulus intensity, we determined acoustic rate-level functions for neurons in the auditory cortex of barbiturate-anesthetized guinea pigs before, during and after administration of ACh. ACh facilitated acoustic rate-level functions in 82% of the cells tested. In addition, during ACh administration 66% of neurons responded to stimuli that were previously subthreshold, that is, ACh decreased the response threshold. Cholinergic facilitation of rate-level functions was attenuated by the general muscarinic antagonist atropine. The nature of the muscarinic receptors involved in the actions of ACh was further examined by presenting single tones before, during, and after administration of ACh and specific muscarinic receptor subtype antagonists, either pirenzepine (M1) or gallamine (M2). ACh-induced facilitation of spontaneous and tone evoked neuronal discharge was antagonized by pirenzepine, but not by gallamine, suggesting the involvement of the M1 muscarinic receptor subtype. These data indicate that ACh can facilitate stimulus-evoked responses and decrease response thresholds for neurons in auditory cortex, possibly via activation of M1 muscarinic receptors. Such effects of ACh acting at muscarinic receptors could underly cholinergic regulation of information processing in the auditory cortex.     

Characterization of [3H]AF-DX 116 binding sites in the rat brain: Evidence for heterogeneity of muscarinic-M2 receptor sites

This study shows that [³H]AF-DX 116 binds specifically, saturably, and with high affinity to putative muscarinic-M2 receptor sites in the rat brain. In homogenates of the hippocampus, cerebral cortex, striatum, thalamus, and cerebellum, [³H]AF-DX 116 appears to bind two subpopulations of muscarinic sites: one class of higher affinity sites (K_d < 4.0 nM) and one class of lower affinity sites (K_d > 50 nM, except in the cerebellum). The apparent maximal capacities (B_max) of [³H]AF-DX 116 sites in forebrain tissues ranged between 34 and 69 fmol/mg protein for the higher affinity site, and between 197 and 451 fmol/mg protein for the lower affinity site. In cerebellar homogenates, the maximal capacity of [³H]AF-DX 116 binding sites was 10.4 ± 0.4 (K_d = 1.9 ± 0.2 nM) and 39.1 ± 2.6 (K_d = 26 ± 7 nM) fmol/mg protein for the higher and the lower affinity site, respectively. Determination of the K_d for the higher and lower affinity [³H]AF-DX 116 sites from association and dissociation constants yielded similar values to those obtained from the saturation data. The ligand selectivity pattern reveals that AF-DX 116 is more potent than (–)QNB > atropine > methoctramine > 4-DAMP > gallamine > NMS > carbamylcholine > oxotremorine > pirenzepine > > nicotine in competing for the higher affinity [³H]AF-DX 116 sites. With few exceptions, the pattern was similar for the lower affinity sites. For example, (–)QNB was more potent than AF-DX 116 and pirenzepine was more potent than either oxotremorine or 4-DAMP at the lower affinity [³H]AF-DX 116 sites. In addition, pirenzepine was modestly more potent at the lower compared to the higher affinity sites. Neither the higher nor the lower affinity [³H]AF-DX 116 sites were sensitive to the effects of Gpp(NH)p or N-ethylmaleimide. In addition, the carbamylcholine-induced inhibition of [³H]AF-DX 116 binding to the higher and the lower affinity sites was altered by Gpp(NH)p and NEM, to a similar extent. However, Gpp(NH)p decreased the affinity of carbamylcholine (i.e., increased the IC₅₀), whereas N-ethylmaleimide had the opposite effect. Furthermore, N-ethylmaleimide also appeared to steepen the curve for the carbamylcholine-induced inhibition of [³H]AF-DX 116 binding, as evidenced by the increased n^H. Thus it appears that [³H]AF-DX 116 binds to two subsets of muscarinic-M2 receptors in the rat brain, which can be differentiated by their affinity for certain agonists and antagonists.     

Muscarinic receptors modulate intracellular calcium level in chick sensory neurons

In the present work we have studied the variation of intracellular calcium levels induced by muscarinic agonists in chick dorsal root ganglia neurons. Muscarinic agonists such as muscarine and oxotremorine cause an increase of intracellular calcium levels in fura-2AM-loaded DRG neurons of E18 chick embryos. This increase was abolished following treatment with 1 microM atropine but not by 1 microM mecamylamine, indicating that the observed intracellular calcium increase, was dependent on muscarinic receptor activation. Stimulation in absence of external calcium or pre-incubation of the DRG cultures with thapsigargin or Mn(2+) demonstrated that [Ca(2+)](i) increase is mainly due to its release from intracellular stores. The use of selective antagonists of muscarinic receptor subtypes also indicated that M(1) and to a lesser extent M(3) receptor subtypes are responsible for the observed intracellular calcium mobilization. Finally pre-treatment of DRG cultures with pertussis toxin showed that the variation of [Ca(2+)](i) levels was dependent on PTX-insensitive G-protein. Moreover muscarinic agonists induce in DRG also the increase of IPs level, suggesting that the variations of intracellular calcium levels may be due at least in part to the activation of the phosphoinositide transduction pathway. In conclusion the reported observations demonstrate the activity of muscarinic receptors in sensory neurons, suggesting a functional role for acetylcholine in sensory transduction.     

Protein phosphorylation in primary astrocyte cultures treated with and without dibutyryl cyclic AMP

Protein phosphorylation was investigated in primary rat astrocyte cultures treated with and without dibutyryl cyclic AMP. Astrocytes maintained in dibutyryl cyclic AMP for several weeks displayed increased phosphate incorporation in 5 protein bands (55, 52, 45, 43 and 28 kDa) while incorporation in one band (42 kDa) was decreased. Phosphate incorporation in several other protein bands was unchanged. Calcium-dependent phosphate incorporation was also altered by prior exposure of the cells to dibutyryl cyclic AMP: addition of calcium to broken cell preparations resulted in increased incorporation in 75, 53 and 52 kDa while decreased incorporation occurred in 100 kDa. These differences in protein phosphorylation may be related to the previously reported biochemical and morphological changes brought about by dibutyryl cyclic AMP and may provide insights into the mechanisms of reactive gliosis.     

Selectivity of pirenzepine in the central nervous system. III. Differential effects of multiple pirenzepine and scopolamine administrations on muscarinic receptors as measured autoradiographically

The effects of intrahippocampal injections of scopolamine and pirenzepine on muscarinic receptor binding were examined by quantitative autoradiographic techniques. Brain slices from animals which had received 7 injections of either scopolamine (n = 5) or pirenzepine (n = 5) over a 22-day injection schedule were compared with slices from 5 saline-injected controls for receptor binding to the whole slice and within selected regions of the brain as measured autoradiographically. The total number of receptors was determined from direct binding assays with 1-[3H]quinuclidinyl-benzilate ([3H]-1-QNB), while the binding of the selective ligands pirenzepine, carbamylcholine, and scopolamine was examined through inhibition studies. The data from the whole slices indicated that pirenzepine-treated animals contained more receptors for [3H]-1-QNB than either saline- or scopolamine-injected controls. Slices from the same animals also displayed a lower affinity for pirenzepine. Slices from scopolamine-injected animals revealed neither an increase in receptor number nor a decrease in antagonist affinity, although the binding of the agonist carbamylcholine was increased. Quantitative analysis of the autoradiograms generated from the slices indicated that the increase in receptor number for pirenzepine-injected animals was predominantly within the cerebral and cingulate cortices. The inhibition by pirenzepine was also lower in these areas in the same group of animals. Agonist inhibition was altered in the central layers of the cerebral cortex and in the pretectal area in scopolamine-treated animals. The results suggest separate mechanisms of drug action and adaptation for pirenzepine and scopolamine.     

Cellular mapping of m2 muscarinic receptors in rat olfactory bulb using an antiserum raised against a cytoplasmic loop peptide

An antiserum that recognizes a sequence from the putative third cytoplasmic loop of the m2 subtype of muscarinic receptors (mAchR) has been raised and used to map the cellular distribution of this subtype in rat olfactory bulb. The antiserum was obtained by injecting BALB/C mice with a BSA-conjugated synthetic peptide whose sequence corresponded to amino acids 240-259 of the porcine cardiac m2 mAChR gene. Antibodies recognized the synthetic peptide in ELISA screening and labelled a single band corresponding to the peak of [3H]PrBCM-labelled heart mAchRs in immunoblots. Immunostaining of olfactory bulb, a region of the brain enriched in this muscarinic receptor subtype, showed that the antibodies labelled cell bodies and multiple dendritic processes. Broad fluorescent labelling throughout cell bodies was consistent with binding to the cytoplasmic face of the surface membrane, in support of the predicted cytoplasmic loop structure. m2-Positive cells throughout the bulb were sparsely distributed in different layers representing small subpopulations of the cells in each region: glomeruli, 6%; external plexiform layer, 16%; inner plexiform and granule cell layer, 3%. The results show that antibodies against specific sequences of different muscarinic receptor subtypes can be used to localize subtypes in situ, that the m2 subtype within the rat olfactory bulb is broadly distributed, and that the m2 subtype can occur postsynaptically in this central nervous system (CNS) region. The mapping of m2-positive cells in olfactory bulb may be of particular interest because loss of this subtype and degeneration of the olfactory system have been observed in Alzheimer's disease.     

Muscarinic receptor-mediated calcium signaling in spiral ganglion neurons of the mammalian cochlea

Using indo-1 microspectrofluorometry, we examined the effects of cholinergic agonists on the concentration of intracellular Ca²⁺ ions ([Ca²⁺]_i) in spiral ganglion neurons, isolated from rat cochleae at different stages of post-natal development (from P3 to P30). Extracellular application of acetylcholine (ACh) or carbamylcholine generated a rapid and transient increase in [Ca²⁺]_i. The ACh concentration-response curve indicated an apparent dissociation constant (K_d) of 8 μM and a Hill coefficient of 1.0. Removing extracellular free Ca²⁺ did not suppress the ACh-induced Ca²⁺ responses suggesting an intracellular Ca²⁺-release mechanism. When we compared the cholinergic response at different stages of postnatal development, there were no significant differences on the aspect of the Ca²⁺ response and the percentage of responsive neurons, which ranged between 50 and 65% per cochlear preparation. The application of muscarine triggered reversible Ca²⁺ responses similar to those observed with ACh, with an apparent K_d of 10 μM and a Hill coefficient of 1.0. The cholinergic-induced Ca²⁺ response was reversibly blocked by muscarinic antagonists with the following order of potency, atropine>4-DAMP>methoctramine>pirenzepine. Nicotine (10 to 100 μM) did not evoke Ca²⁺ responses and the nicotinic antagonist curare (10 μM) did not block the ACh-evoked responses. The present study is the first direct demonstration of functional muscarinic receptors (mAChRs) in spiral ganglion neurons. These mAChRs activated by the cholinergic lateral efferent system may participate in the regulation of the electrical activity of the afferent auditory fibers contacting the inner hair cells.     

Biochemical and behavioral responses of pilocarpine at muscarinic receptor subtypes in the CNS. Comparison with receptor binding and low-energy conformations

Pilocarpine was tested biochemically in vitro for its ability to stimulate phosphoinositide (PI) turnover in the hippocampus (M1/M3 responses) where it displayed 35% of the maximal carbachol response with an EC50 value of 18 microM, and low-Km GTPase in the cortex (M2 response), where it had 50% of the maximal carbachol response with an EC50 value of 4.5 microM. Behaviorally, pilocarpine was able to restore deficits in a representational memory task (sensitive to M1 antagonists) produced by intrahippocampal injections of AF64A. Twenty-three low-energy conformations of protonated pilocarpine were generated using the program MacroModel. The data indicate that pilocarpine is a partial agonist at both M1 and M2 muscarinic receptors in the CNS. Behaviorally, with respect to the memory task, M1 effects of pilocarpine apparently predominate. It also is conceivable that different conformations of pilocarpine are active as agonists at different muscarinic receptor subtypes.     

Receptor reserve of phosphoinositide-coupled muscarinic receptors in mouse hippocampus in vivo

The ability of the partial muscarinic agonist pilocarpine to increase in vivo phosphoinositide (PI) hydrolysis in mouse brain was compared to two full agonists. Pilocarpine increased in vivo phosphoinositide (PI) hydrolysis in cortex, striatum, and to the greatest extent in the hippocampus. Pilocarpine injected either subcutaneously or intracerebroventricularly robustly increased in vivo PI hydrolysis in hippocampus up to 500% of control levels and the increases were blocked by the muscarinic antagonist scopolamine. The increases in vivo PI hydrolysis induced by pilocarpine were 60-75% of the magnitude of the full muscarinic agonists oxotremorine-M and cis-dioxolane. The muscarinic M(1) preferring antagonist pirenzepine potently blocked pilocarpine-induced increases in in vivo PI hydrolysis, consistent with the increase being mediated by M(1) receptors. Since pilocarpine is a relatively weak partial agonist, these data suggest a substantial level of receptor reserve for the PI response in mouse hippocampus.     

Stimulation of phosphoinositide hydrolysis in myelin by muscarinic agonist and potassium

Slices of rat brainstem that had been prelabeled by in vivo injection of [3H]inositol were stimulated with carbachol in the presence of lithium and changes measured in the radioactivity of inositol lipids and water-soluble inositol phosphates. For the latter, significant increases were seen for inositol mono- and bisphosphate but not inositol trisphosphate. Analysis of whole tissue phosphoinositides revealed significantly reduced radioactivity in phosphatidylinositol and phosphatidylinositol 4-phosphate, whereas myelin showed decreases in those as well as phosphatidylinositol 4,5-bisphosphate. These effects were blocked by atropine. Stimulation of the tissue slices with elevated K+ resulted in increased formation of inositol phosphate and decreased radioactivity in phosphatidylinositol. The effect was not blocked by atropine and in the presence of this agent, which reduced background reaction, all 3 phosphoinositides showed significant K+-induced loss of label. Elevated K+ and carbachol thus function through different mechanisms in this system. Carbachol is believed to affect myelin phosphoinositides through direct interaction with muscarinic receptors which were recently shown to be present in this membrane.     

Characterization of dopamine receptors on neurons grown in primary dissociated cell culture from ventral mesencephalon of mouse

Mammalian neurons from ventral mesencephalon were grown in primary dissociated cell culture. These cultures were examined for dopamine sensitive adenylate cyclase activity and specific ligand binding of [3H]spiroperidol and [3H]flupenthixol. No stimulation of adenylate cyclase activity by 10 microM dopamine was demonstrable in cell culture homogenates. [3H]Spiroperidol bound to cell culture homogenates with high affinity and was displaced by (+)-butaclamol but not by 5-hydroxytryptamine, suggesting that the [3H]spiroperidol was bound to dopamine receptors. While [3H]flupenthixol binding was also present, it could be displaced by spiroperidol indicating that the dopamine receptor was probably of the D2 subtype. Binding of spiroperidol was proportional to the amount of cell culture homogenate, and was saturable. Are these receptors autoreceptors? The toxin 1-methyl-4-phenylpyridine (MPP+) was used to destroy dopaminergic neurons; spiroperidol binding in these cultures was found to be increased, demonstrating that most of these D2 receptors are not autoreceptors.