Loss of muscarinic M4 receptors in hippocampus of Alzheimer patients

We assessed muscarinic M₁, M₂ and M₄ receptor subtypes in the hippocampus of Alzheimer’s and control brains by receptor autoradiography using ligands such as [¹²⁵I]muscarinic toxin-1 ([¹²⁵I]MT-1, M₁ selective), [³H]AFDX-384 (M₂ partially selective) and [¹²⁵I]muscarinic toxin 4 ([¹²⁵I]M₄ toxin-1, M₄ selective). Our results revealed a significant decrease in muscarinic M₄ receptor binding in the dentate gyrus and CA4 regions of brain sections from Alzheimer’s patients compared to controls. No changes in the density of M₁ or M₂ receptor binding were observed. Our findings suggest that, relative to other muscarinic receptor subtypes, the M₄ receptor could be the subtype which is selectively compromised in Alzeheimer’s disease (AD).     

Ontogeny of muscarinic receptors in the rat brain with emphasis on the differentiation of M1- and M2-subtypes— semi-quantitative in vitro autoradiography

The ontogeny of muscarinic acetylcholine receptors (mAChR) in the rat brain was studied with emphasis on the differentiation of M₁- and M₂-receptor subtypes through semi-quantitative in vitro autoradiography. [³Quinuclidinyl benzilate] (QNB) and [³H]pirenzepine (PZ) were used for labeling total mAChR and M₁-receptors, respectively. In the cerebral cortex of adult rats, [³H]QNB binding sites were more richly present in the superficial and deeper layers than in the middle layer, while M₁-receptors were diffusely observed in all the layers. This means that M₂-receptors are highly concentrated in the superficial and deeper layers. The ontogenetical differentiation of the laminar distribution between M₁- and M₂-receptor first appeared at 14 days of postnatal age. In the hippocampus and striatum whose mAChR were predominantly of the M₁-type in the adult rat brain, ontogenic patterns of M₁-receptors were almost identical to those of total mAChR. On the other hand, mAChR in the cerebellar cortex and lower brainstem of the adult rat were mainly of the M₂-subtype. In these areas, the ontogeny of total mAChR was apparently observed. However, M₁-receptors were not observed at any stage of the ontogeny. The above-mentioned results indicate that M₁- and M₂-receptors show distinct developmental behaviors in the rat brain.     

Functional and biochemical basis for multiple muscarinic acetylcholine receptors

1. The novel antimuscarinic compound pirenzepine (PZ) has generated considerable interest in the basis and the implications of muscarinic acetylcholine receptor (mAChR) heterogeneity.

2. [³H]PZ has been used extensively to identify and characterize the putative M₁ (high affinity for PZ) mAChR subtype, which predominates in central nervous system (CNS) and ganglia.

3. The heterogeneity sensed by PZ is not identical to the heterogeneity sensed by agonists.

4. Differences in effector coupling do not necessarily provide a simple explanation for the molecular basis of these putative M₁ and M₂ subtypes.

5. Therapeutic and untoward effects of muscarinic drugs may be mediated by independent mAChR subpopulations which may be pharmacologically exploited to produce more highly selective as well as efficacious new drugs.     

5-HT1B receptor binding in degenerative movement disorders

Using [³H]sumatriptan as a radioligand, 5-hydroxytryptamine (5-HT)_1B receptors were examined in posterior striatum and midbrain post-mortem tissue sections of 12 patients who had died from representative degenerative movement disorders as compared to nine controls. In the control human basal ganglia, the highest densities of [³H]sumatriptan binding were observed in the globus pallidus and substantia nigra. No significant change in the density of [³H]sumatriptan binding sites was found in the striatum and substantia nigra of the six Parkinson's disease brains. In the two brains from patients with progressive supranuclear palsy an increase was found in the densities of [³H]sumatriptan binding sites, most marked in the substantia nigra. In contrast, [³H]sumatriptan labelling was almost absent in the striatonigral degeneration brain and was markedly reduced in the three Huntington's disease brains. This study indicates that the status of 5-HT_1B receptors is different in each degenerative movement disorder and suggests that human 5-HT_1B receptors are located somatodendritically on GABAergic and peptidergic caudate-putamen neurons which project to the substantia nigra and globus pallidus, where these receptors are presynaptic.     

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.     

Effect of Chronic Administration of Haloperidol (Intermittently) and Haloperidol-Decanoate (Continuously) on D2 Dopamine and Muscarinic Cholinergic Receptors and on Carbachol-Stimulated Phosphoinositide Hydrolysis in the Rat Striatum

Abstract: It has been reported that apomorphine-induced stereotypy is sensitized after a chronic intermittent administration of haloperidol (HPD), but not after a chronic continuous exposure to haloperidol-decanoate (HPD-D). The present study was undertaken to investigate changes in the D₂ dopamine and muscarinic receptors in the ratstriatum after the administration of HPD intermittently and HPD-D continuously. The number of striatal [³H] spiperone binding sites increased significantly after HPD-D, but did not change after HPD. Neither the number of [³H](–)QNB binding sites nor carbachol-stimulated phosphoinositide hydrolysis changed after either HPD or HPD-D. These results indicate that the increase in striatal D₂ receptors in rats administered HPD-D represents behavioral and biochemical tolerance, and that neither the D₂ dopamine receptor supersensitivity nor muscarinic receptor hyposensitivity underlies sensitization of apomorphine-induced stereotypy.     

Pirenzepine-insensitive muscarinic autoreceptors regulate acetylcholine release in human neocortex

The release of [³H]acetylcholine ([³H]ACh) and its modulation mediated by autoreceptors were investigated in synaptosomes prepared from fresh human cerebral cortex prelabelled with [³H]choline ([³H]Ch) and depolarized in superfusion with 15 mM KCl. The K⁺-evoked release of tritium was almost totally accounted for by unmetabolized [³H]ACh and was largely calcium-dependent. Exogenous ACh decreased the depolarization-evoked release of [³H]ACh in a concentration-dependent manner (EC₅₀ = 1.5 μM). The inhibitory effect of ACh on [³H]ACh release was counteracted by the non-selective muscarinic antagonist atropine. In contrast, the selective M₁ receptor antagonist pirenzepine was ineffective. It is concluded that muscarinic autoreceptors regulating the release of ACh are present on cholinergic nerve terminals of human cerebral cortex and appear to belong to a pirenzepine-insensitive subtype.     

Denervation supersensitivity for benzodiazepine receptors in the rat substantia nigra

The injection of kainic acid into the substantia nigra causes, 3 weeks after treatment, a 40% decrease in the total number of binding sites for [³H]diazepam with an increase in the dissociation constant. This decrease was restored to approximately normal by the subsequent injection of kainic acid into the striatum, homolateral to the lesioned substantia nigra. The injection of kainic acid into the striatum of intact animals failed to modify the number of [³H]diazepam binding sites but increased the K_D. The results indicate that benzodiazepine binding sites in the substantia nigra are partly located on kainic acid sensitive elements (probably interneurons) and, partly, on kainic acid resistant ones. These binding sites become supersensitive after degeneration of striato-nigral pathways. The possible role of GABAergic denervation of the substantia nigra in the development of benzodiazepine binding sites supersensitivity is discussed.     

Alterations in neurotransmitter receptors and glucose use after unilateral orbital enucleation

Serotonin (5-hydroxytryptamine; 5-HT), acetylcholine and γ-aminobutyric acid (GABA) are neurotransmitters in the rat visual system. Using quantitative autoradiography, the effect of unilateral orbitral enucleationon [^3H]5-HT, [³H]ketanserin, [³H]quinuclidinyl benilate (QNB) and [³H]muscimol binding to 5-HT₁, 5-HT₂, muscarinic and GABA_A receptors has been examined within anatomical components of the visual pathway at 4 time points up to 20 days after the lesion. The functional deficit was assessed in the same animals using quantitative [¹⁴C]2-deoxyglucose autoradiography. At 1 day after unilateral orbital enucleation, there were no significant alterations in ligand binding although local cerebral glucose use was reduced in primary visual structures in the visually deprived hemisphere. At 5 days post-enucleation, however, [³H]5-HT binding was significantly reduced in both the visually deprived superior colliculus (by 17%) and dorsal lateral geniculate body (DLG) (by 33%). There were similar alterations in the binding of this ligand in these primary retinal projections areas at 10 and 20 days after orbital enucleation, but there were no changes in secondary areas (e.g. visual cortex) at any time point. [³H]Muscimol binding was significantly reduced in the visually deprived DLG (30%) and visual cortex (21%) only at 20 days post-lesion, whilst [³H]ketanserin and [³H]QNB were not altered in any region in the visually deprived hemisphere at any time point post-enucleation. At 10 and 20 days post-enucleation, the degree of [³H]5-HT, and [³H]muscimol binding deficits in visually deprived structures correlated significantly with the level of reduced metabolic activity in these areas (r = 0.700andr = 0.543respectively). The specificity and regional and temporal heterogeneity of neurotransmitter receptor binding alterations provides evidence of selective adjustments within visual system component6s in response to orbital enucleation.     

A selective lesion of striatonigral neurons decreases presynaptic binding of [H]hemicholinium-3 to striatal interneurons

We have used the suicide transport agent, volkensin, to produce selective lesions of striatal efferent neurons projecting to the substantia nigra in the rat. In order to evaluate potential trans-synaptic effects, we examined cholinergic interneurons intrinsic to the striatum following destruction of striatonigral projection neurons by nigral injection of volkensin. There was no change in the number of large interneurons identified either by Nissl stain or by immunocytochemistry for choline acetyltransferase, indicating that volkensin was not directly toxic to this group of neurons. However, [³H]hemicholinium-3 binding to the choline re-uptake site on the presynaptic cholinergic terminals decreased. No change in [³H]hemicholinium-3 binding was seen after destruction of dopaminergic afferents with 6-hydroxydopamine. Striatonigral afferents to the cholinergic interneurons contain substance P which has been shown to stimulate acetylcholine release. The decrease in [³H]hemicholinium-3 binding may reflect loss of this afferent input. However, striatonigral neurons are an efferent target of the cholinergic interneuron as well, and a presynaptic effect due to loss of target neurons also may contribute.     

Lasting increase in D1 dopamine receptors in the lateral part of the substantia nigra pars reticulata after subchronic methamphetamine administration

To examine the possible involvement of D₁ dopamine receptors in behavioral sensitization induced by subchronic methamphetamine (MAP) administration, regional D₁ receptors labeled with [³H]SCH 23390 were examined using binding assay and quantitative autoradiography. Rats received 4 mg/kg/day MAP (i.p.) for 14 days, and were decapitated after an abstinence period of 24 h, 7 days or 21 days. In MAP-treated rats, a significant decrease in K_d in the mesolimbic area was observed 24 h but not 7 days after the last injection. Neither K_d nor B_max changed in the striatum or medial prefrontal cortex of MAP-treated rats after any period of abstinence. Autoradiography revealed a significant increase in specific [³H]SCH 22390 binding in the lateral part of the substantia nigra pars reticulata (SNr) of MAP-treated rats. Since this increase lasted up to 21 days after cessation of subchronic MAP administration, it is suggested that lasting increase in the nigral D₁ receptors may be associated with the biological changes underlying MAP-induced behavioral sensitization.     

δ1-OPIOID receptor-mediated controlofacetylcholine (ACh) release in human neocortex slices

In slices of human neocortex, prelabelled with [³H]-choline, the release of [³H]-acetylcholine reflects the evoked release of endogenous acetylcholine whichwaselicited by the same electrical stimulation paradigm. [³H]-Acetylcholine releasewasdepressed by the δ-opioid receptor agonist -Pen₂- -Pen₅-enkephalin. When the nerve endings were depolarized byelevatingextracellular potassium the evoked [³H]-acetylcholine release was similarlydepressed by -Pen₂- -Pen₅-enkephalin in theabsence, but notin the presence, of tetrodotoxin which blocks action potential propagation.Therefore, the δ-opioid receptor inhibiting [³H]-acetylcholine release should notbe located tocholinergic nerve terminals, but rather to interneurons. The somatostatin₂receptorpartial agonist octreotide per se did not influence action potential-evoked [³H]-acetylcholine release, but prevented the inhibition of release of [³H]-acetylcholine by -Pen₂- -Pen₅-enkephalin.Similarly, the δ₁-opioid receptor antagonist 7-benzylidenenaltrexon perse did notinfluence [³H]-acetylcholine release, but prevented of the inhibition ofrelease by -Pen₂- -Pen₅-enkephalin.

From the presentfindings we conclude : (1) The evoked release of [³H]-acetylcholine from humanneocortex slices reflects the release of endogenous acetylcholine.(2) It is inhibited in an indirectmanner by opioid receptors of the δ₁-subtype,which (3) are not localized oncholinergic axon terminals but on soma and dendrites ofsomatostatin-containing interneurons,where they inhibit somatostatin release. (4) Theseinterneurons innervate cholinergic nerveendings in the human neocortex and appear to facilitateacetylcholine release via somatostatin₂ receptors.     

Rats bred for enhanced apomorphine susceptibility have elevated tyrosine hydroxylase mRNA and dopamine D2-receptor binding sites in nigrostriatal and tuberoinfundibular dopamine systems

From a Wistar population two rat lines were generated using as criterion the behavioral response to the dopamine agonist apomorphine. Rats of the apomorphine-susceptible (apo-sus) line revealed a vigorous gnawing response to apomorphine administration while the other rat line, the apomorphine-unsusceptible (apo-unsus) line, was selected for lack of response to the drug. In the present study using the 12th and 13th generation of these genetically selected lines, we have investigated whether this difference in apomorphine responsiveness was correlated with changes in dopamine neurochemistry. Therefore, we measured tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis, as well as dopamine D₁ and D₂ receptor mRNA levels in discrete brain regions by in situ hybridization. Dopamine (D₂/D₃) receptor binding was assessed with [¹²⁵I]iodosulpride in a membrane binding assay and by quantitative autoradiography on tissue sections. [³H]SCH 23390 was used to analyze D₁ receptor binding. Apo-sus rats displayed significantly higher TH mRNA levels in the A₉ cell group of the substantia nigra pars compacta and in the A₁₂ cell group of the arcuate nucleus. No difference was found in the A₁₀ cell group of the VTA and the A₆ cell group of the locus coeruleus. The density ofD_2/3 binding sites as well as D₁ receptor mRNA levels in the striatal projection area of the A₉ substantia nigra neurons, were significantly elevated in apo-sus rats. Dopamine D₂ receptor mRNA and D₁ receptor binding levels in caudate putamen and nucleus accumbens, however, were similar in rats of both lines. In conclusion, high apomorphine susceptibility is related to a potentially enhanced dopamine responsiveness selective for the nigrostriatal and tuberoinfundibular pathways.     

Dopamine D2 receptor-mediated G-protein activation in rat striatum: functional autoradiography and influence of unilateral 6-hydroxydopamine lesions of the substantia nigra.

Unilateral 6-hydroxydopamine (6-OHDA) lesions of substantia nigra pars compacta (SNPC) neurons in rats induce behavioural hypersensitivity to dopaminergic agonists. However, the role of specific dopamine receptors is unclear, and potential alterations in their transduction mechanisms remain to be evaluated. The present study addressed these issues employing the dopaminergic agonist, quinelorane, which efficaciously stimulated G-protein activation (as assessed by [³⁵S]GTPγS binding) at cloned hD₂ (and hD₃) receptors. At rat striatal membranes, dopamine stimulated [³⁵S]GTPγS binding by 1.9-fold over basal, but its actions were only partially reversed by the selective D₂/D₃ receptor antagonist, raclopride, indicating the involvement of other receptor subtypes. In contrast, quinelorane-induced stimulation (48% of the effect of dopamine) was abolished by raclopride, and by the D₂ receptor antagonist, L741,626. Further, novel antagonists selective for D₃ and D₄ receptors, S33084 and S18126, respectively, blocked the actions of quinelorane at concentrations corresponding to their affinities for D₂ receptors. Quinelorane potently induced contralateral rotation in unilaterally 6-OHDA-lesioned rats, an effect abolished by raclopride and L741,626, but not by D₃ and D₄ receptor-selective doses of S33084 and S18126, respectively. In functional ([³⁵S]GTPγS) autoradiography experiments, quinelorane stimulated G-protein activation in caudate putamen and, to a lesser extent, in nucleus accumbens and cingulate cortex of naive rats. In unilaterally SNPC-lesioned rats, quinelorane-induced G-protein activation in the caudate putamen on the non-lesioned side was similar to that seen in naive animals (50% stimulation), but significantly greater on the lesioned side (80%). This increase was both pharmacologically and regionally specific since it was reversed by raclopride, and was not observed in nucleus accumbens or cingulate cortex. In conclusion, the present data indicate that, in rat striatum, the actions of quinelorane are mediated primarily by D₂ receptors, and suggest that behavioural hypersensitivity to this agonist, induced by unilateral SNPC lesions, is associated with an increase in D₂, but not D₃ or D₄, receptor-mediated G-protein activation.     

Autoradiographic distribution of binding sites for the non-NMDA receptor antagonist [H]CNQX in human motor cortex, brainstem and spinal cord

The distribution of non-NMDA receptors in the normal human motor cortex, brainstem and spinal cord has been investigated using [³H]CNQX. In the motor and premotor cortex, specific [³H]CNQX binding was present in all cortical laminae with the highest density of binding sites in laminae I, II and the upper part of III. In the normal brainstem, non-NMDA receptors labelled by [³H]CNQX had a heterogenous distribution. Brainstem motor nuclei subserving eye movements, which tend to be spared in motor neuron disease (MND), had a higher density of [³H]CNQX binding sites compared to other cranial nerve motor nuclei (VII, X, XII) which tend to be affected. Specific [³H]CNQX binding was present throughout the spinal grey matter, the greatest density of binding being found in the substantia gelatinosa. Excitotoxicity at non-NMDA receptors has been implicated in chronic neurodegenerative diseases such as motor neuron disease. This study suggests that the density of non-NMDA receptors, labelled by [³H]CNQX, does not account for selective vulnerability of motor neurons in this disorder.     

Muscarinic cholinergic receptors in the human spinal cord: differential localization of [H]pirenzepine and [H]quinuclidinylbenzilate binding sites

The localization of muscarinic cholinergic receptor subtypes was studied in the human spinal cord using in vitro labelling of cryostat sections with [³H]quinuclidinylbenzilate (QNB) and [³H]pirenzepine (PZ) followed by autoradiography. The highest densities of [³H]QNB binding were localized in laminae II (substantia gelatinosa) and IX (motor neurons); in contrast, the highest density of [³H]PZ binding was localized to lamina II where the binding density was 22—32% higher than in lamina IX. These results suggest that the M₁ and M₂ muscarinic cholinergic receptor subtypes may be differentially localized in sensory and motor regions of the human spinal cord.     

Transferrin receptors in the Parkinsonian midbrain

Several hypotheses have been put forward to explain the pathogenesis of Parkinson's disease (PD) and recently it has been suggested that alterations in iron homeostasis may be implicated. Because of the central role of the transferrin receptor in providing access of iron to cells, we have studied the distribution and density of transferrin receptors using [³H]–transferrin ([³H]–Tf) binding and tritium film autoradiography in the normal and PD midbrain. High levels of [³H]–Tf binding were found in the dorsal raphé, oculomotor nucleus and periaqueduc tal grey whilst lower levels of [³H]–Tf binding were found in the tegmentum, red nucleus and substantia nigra. Significant reductions in binding were found in the substantia nigra, red nucleus and oculomotor nucleus in PD, the reductions in [³H]–Tf binding being similar to the loss of nigral neurons in PD. The data suggest that the increased iron content of surviving nigral neurons may reflect a compensatory metabolic response rather than abnormal transferrin receptor expression.     

Properties of the phencyclidine (PCP) receptors

1. 1. [³H]Phencyclidine (PCP, Angel Dust) receptors have been characterized using a rat brain binding section technique.

2. 2. [³H]PCP labels a single class of site in rat brain (k_d = 46 nM; B_max = 10.5 fold/slice). Ligand selectivity pattern strongly suggests that [³h]pcp binds to sites relevant for its pharmacological actions.

3. 3. Chronic PCP treatment (10 mg/kg/day for 14 days) decreases the number of sites (B_max) for [³H]PCP and [³H]spiperone binding but not for [³H]dihydromorphine. These modifications could be related to the development of tolerance and dependence to PCP.

4. 4. Visualization of [³H]PCP binding sites shows high densities of receptors in cortical areas and hyppocampus. Lower densities are observed in caudate-putamen, nucleus accumbens, and amygdala. Negligible quantities of receptors are seen in brain stem and over white matter.

5. 5. The presence of specific [³h]pcp binding sites in rat brain suggests the possible existence of an endogenous ligand for this unique receptor.

Effect of electroconvulsive shock on central cholinergic (muscarinic) receptors

Sensitivity of central cholinergic (muscarinic) receptors was studied in rats after application of single and repeated (once daily for 7 days) electroconvulsive shocks (ECS). Oxotremorine-induced tremor was significantly enhanced after single as well as chronic exposure to ECS; [³H]QNB binding in frontal cortex was also significantly increased by chronic ECS. The results indicate that ECS develops supersensitivity in central muscarinic receptors.     

Impaired axonal transport of opiate and muscarinic receptors in streptozocin-diabetic rats

Axonal transport of receptors was studied in streptozocin-diabetic rats using two different binding models. Streptozocin-induced hyperglycemia caused a reduced accumulation of muscarinic receptors above a ligature placed on rat sciatic nerves when the binding assay was performed in vitro with [³H]QNB. In the vagus nerve, the retrograde axonal transport of receptor-bound opiate was strongly decreased in the streptozocin-treated rats when [³H]lofentanil was used in vivo to label opiate receptors. Insulin partly reversed the changes observed in the streptozocin-treated rats. These findings suggest that impaired axonal transport of receptors may explain part of the neurological disturbance which is seen in diabetic patients.