Autoradiographic distribution of forskolin and phorbol ester binding sites in the retina

We have localized the distribution of [3H]forskolin and [3H]phorbol dibutyrate binding sites autoradiographically in the rat, monkey, and human retina. In the rat and monkey retina, forskolin binding was enriched in the inner plexiform layer, in the inner and outer segments of the photoreceptors, and in the retinal pigment epithelium. In the human retina, forskolin binding sites were uniformly distributed and higher in density. Forskolin binding was also detected over the ciliary body, the ciliary epithelium, and the iris sphincter. The distribution of phorbol ester binding sites was similar in the rat, monkey, and human retina. The inner plexiform layer contained the highest density followed by the inner nuclear and outer plexiform layers, and the ganglion cell layer. In the rat, phorbol ester binding was present in the iris, the ciliary body, and the ciliary epithelium. The monkey and human ciliary body also contained a low density of phorbol ester binding sites.     

Specific [H]strychnine binding associated with glycine receptors in bovine retina

Saturable, specific [³H]strychnine binding can be demonstrated in homogenates of bovine retina. Scatchard plots revealed only one set of binding sites with a dissociation constant (K_d) of about 60 nM and a maximal number of binding sites of about 1.5 pmol/mg protein. The structural specificity of [³H]strychnine binding sites in bovine retina parallels the properties found for [³H]strychnine binding sites in the spinal cord of several vertebrates. Thus, the data do not give any evidence that specific [³H]strychnine binding in bovine retina labels taurine rather than glycine receptors and favors glycine rather than taurine as inhibitory neurotransmitter in bovine retina. The subcellular distribution of specific [³H]strychnine binding in bovine retina parallels that of sodium-dependent, high-affinity uptake of glycine and taurine. All 3 parameters are mainly found in the P₂ fractions of bovine retina homogenates, containing conventional synaptosomes, most abundant in the inner plexiform layer, but can also be found in the P₁ fractions, containing large synaptosomes from the photoreceptor cell layer.     

Identification and localization of adrenergic receptors in cat visual cortex

The concentration and location of adrenergic receptors in cat visual cortex have been determined by radioligand binding techniques using [3H]prazosin (alpha 1-adrenergic receptors), [3H]yohimbine (alpha 2-adrenergic receptors) and [3H]dihydroalprenolol (beta-adrenergic receptors). Saturable high affinity binding sites for all of these ligands were found. The beta-adrenergic receptor population was resolved into beta 1- and beta 2-sites that were present in the ratio 35:65. The laminar distributions of the alpha 1-, alpha 2- and beta-adrenergic receptors were different. The alpha 1- and beta-adrenergic receptors were very similarly localized, being seen in upper layers (I, II and III) and lower layers (layers V and VI). The labelling in upper layers was greater than that in lower layers, more so for alpha 1-adrenergic receptors than beta-adrenergic receptors. alpha 2-Adrenergic receptors were seen in a single band that occupied layer II and III but did extend to the pial surface. These results indicate that the effect of norepinephrine on neuronal activity in cat visual cortex will depend upon the layer in which it is released. Our results provide a basis for further physiological studies of the role of norepinephrine in the processing of visual information.     

Strychnine-insensitive [H] glycine binding to synaptosomal membranes from the chick retina

The pharmacology and kinetics of strychnine-insensitive [[3]H] glycine binding to synaptic membranes from the outer (P₁) and the inner (P₂) plexiform layers of chick retina was studied. Inhibition curves for glycine, d-serine, 1-amincyclopropanecarboxylic acid (ACPC) and strychnine were analyzed by non-linear regression. Hill slopes for glycine and d-serine were not different from unity, whereas those for ACPC were < 1 in both fractions, revealing heterogeneity of binding sites in these membranes. Non-linear regression analysis of time course and saturation experiments strengthen the idea that [[3]H] glycine binds to more than one class of sites, with similar affinities at equilibrium. Antagonists of strychnine-insensitive glycine receptors in the CNS did not inhibit [[3]H] glycine binding to these membranes, which demonstrates that NMDA receptors in the retina have different structural requirements for ligand interaction at these sites. pH affected the specific binding, in agreement with the participation of specific amino acid residues at glycine binding sites on NMDA receptors, and also with functional studies in which the modulation of affinity at this site by protons has been observed. These results support previous studies regarding CPP and MK-801 binding, and provide evidence which indicates that the pharmacophore for glycine and other NMDA-related ligands is distinct for the retina, compared to the CNS, mainly regarding the effects of glycine-site antagonists.     

Localization and characterization of somatostatin binding sites in the mouse retina

We studied the binding of [125I]Tyr11-somatostatin-14 and [125I]Leu8,D-Trp22,Tyr25-somatostatin-28 to frozen, unfixed sections of C57BL/6J mouse eyes with autoradiography. Specific binding of both ligands occurred in 3 maxima, a broad band extending from the retinal ganglion cell to the inner nuclear layers, a narrow and inconstant band over the outer plexiform layer, and a band over the retinal pigment epithelium and choroid. We quantified the label over the inner plexiform layer and found evidence for a single, saturable binding site after Scatchard analysis of saturation binding data. With [125I]Tyr11-somatostatin-14 the dissociation constant (Kd) was 1.48 nM and the total number of binding sites (Bmax) was 68 fmol/mg protein; in competition experiments the inhibitory binding constant (Ki) was 900 pM for somatostatin-14 and 350 pM for somatostatin-28. With [125I]Leu8,D-Trp22,Tyr25-somatostatin-28, Kd was 625 pM and Bmax was 69 fmol/mg protein; in competition experiments Ki was 4.58 nM for somatostatin-14 and 710 pM for somatostatin-28. These results demonstrate the existence of somatostatin receptors in the inner plexiform layer of the retina that appear to have greater specificity for somatostatin-28 than for somatostatin-14.     

Anatomy of brain alpha 1-adrenergic receptors: in vitro autoradiography with [125I]-heat

Much useful information on the localization of alpha 1-adrenergic binding sites has been gained by using tritiated radioligands for in vitro autoradiography. However, the iodinated alpha 1-adrenergic antagonist HEAT [( 2-beta (4-hydroxyphenyl)-ethylaminomethyl)-tetralone], BE 2254), a radioligand with high affinity and specificity, provides autoradiographs with a higher signal to noise ratio. This has allowed us to describe the anatomy of these binding sites in much greater detail than previously possible. Regions showing the highest levels of binding include external plexiform layer of the olfactory bulb, layers Va and Vc of frontoparietal cortex, lateral and central amygdaloid nuclei, thalamus, and inferior olive. Other regions were generally less intensely labeled, with the least evidence of labeling in white matter, such as corpus callosum. Some regions (e.g., hippocampus) had only moderate labeling, but the binding appeared in a discrete pattern that reflected the functional organization of the structure. Although the [125I]-HEAT binding sites were distributed in a pattern similar to that previously reported for [3H]-WB 4101 and [3H]-prazosin, the anatomical detail seen with the iodinated ligand is greater. As a result, an association of alpha 1-adrenergic antagonist binding sites with specific layers in the cortex and with some catecholamine-containing nuclei in the brainstem, such as the locus coeruleus, have been seen for the first time.     

Interaction between alpha 2- and beta-adrenergic receptors in rat cerebral cortical membranes: clonidine-induced reduction in agonist and antagonist affinity for beta-adrenergic receptors.

The interaction between alpha 2- and beta-adrenergic receptors was investigated in rat cerebral cortical membranes. Clonidine inhibition of [3H]dihydroalprenolol ([3H]DHA) binding resulted in biphasic competition curves with a mean Hill coefficient of 0.45. The addition of 1 microM yohimbine caused a rightward shift of the first portion of the clonidine inhibition curve. In the presence of 1 microM clonidine, the maximum concentration which did not inhibit [3H]DHA binding, inhibition curves of [3H]DHA binding by isoproterenol shifted to the right. A mean Hill coefficient increased from a control value of 0.63 to 0.76. Computer modeling analysis revealed that 1 microM clonidine decreased a beta-adrenergic high-affinity state from 28% to 13%. However, the addition of 1 microM yohimbine completely prevented the clonidine-induced reduction in the beta-adrenergic high-affinity state. In the presence of 200 microM GTP, the effect of clonidine was not observed. In addition, Kd and Bmax values for [3H]p-aminoclonidine ([3H]PAC) binding were not significantly changed by the addition of 100 nM isoproterenol, the maximum concentration which did not inhibit [3H]PAC binding. Moreover, isoproterenol inhibition of [3H]PAC binding resulted in steep competition curves with a mean Hill coefficient of 0.97. The addition of 1 microM alprenolol did not affect the isoproterenol inhibition curve. These data demonstrated that clonidine caused a decrease in agonist and antagonist affinity for beta-adrenergic receptors, while isoproterenol did not modulate the binding characteristics of alpha 2-adrenergic receptors. Furthermore, these results suggest that regulation between alpha 2- and beta-adrenergic receptors is not bidirectional, but is instead unidirectional from alpha 2-adrenergic receptors to beta-adrenergic receptors.     

Developmental regulation of adenosine A1 receptors, uptake sites and endogenous adenosine in the chick retina.

Although adenosine A1 receptors mediate the inhibition of dopamine-dependent stimulation of adenylate cyclase activity in the developing chick retina, their localization and function are unknown. We have examined the localization of these receptors, and of endogenous adenosine and adenosine uptake sites at several stages of chick retinal development. A1 receptors were already localized predominantly to plexiform regions by embryonic day 12 (E12) with no gross changes at subsequent stages. Adenosine immunoreactivity was absent from retina at E8 but was detected at E12 in the ganglion cell layer, as well as cells in the inner nuclear cell layer and photoreceptors. At more advanced developmental stages the immunoreactivity was greater, but displayed similar localizations. Uptake sites labeled with [3H]nitrobenzylthioinosine (NBI) were detected even earlier using binding and autoradiographic methods. [3H]NBI binding was saturable, and Scatchard analysis demonstrated a single class of sites with a Kd of 0.91 nM and Bmax of 298 fmol/mg protein in E15 retinal membranes. The binding was displaced by unlabeled NBI and dipyridamole. NBI binding sites differentiated earlier than adenosine A1 receptors or endogenous adenosine immunoreactivity, showing a diffuse distribution at E8, but predominating in the plexiform layers of more developed retinas. The results indicate that elements of a putative purinergic system differentiate at specific localizations early in retinal development.     

Modulation of rat brain alpha- and beta-adrenergic receptor populations by lesion of the dorsal noradrenergic bundle

Bilateral lesion of the ascending noradrenergic fibers in the dorsal bundle of adult Wistar rats with 4 micrograms 6-hydroxydopamine caused extensive depletion of norepinephrine in all forebrain areas, but led to a 54% increase in norepinephrine levels in the cerebellum. beta-Adrenergic receptor binding of [3H]dihydroalprenolol was significantly increased in all forebrain areas depleted of norepinephrine except hypothalamus. The increase in [3H]dihydroalprenolol binding was due to 62% and 34% increases in the number of beta-receptor sites in the frontal cerebral cortex and hippocampus respectively. Binding of [3H]WB-4101 to alpha 1-adrenergic receptors after dorsal bundle lesion was augmented generally to a lesser extent than beta-receptor binding, with significantly increased numbers of sites only in the frontal cortex (74%), thalamus (20%) and septum. Both alpha 1- and beta-receptor binding sites were reduced in number by 25-28% in the cerebellum of dorsal bundle-lesioned rats, whereas intraventricular administration of 6-hydroxydopamine to adult rats, which depletes norepinephrine in the cerebellum by 96%, increased cerebellar alpha 1- and beta-receptor binding by 33-40%. Binding of [3H]clonidine to forebrain alpha 2-adrenergic receptors was significantly elevated in the frontal cortex, but reduced in the amygdala and septum, after dorsal bundle lesion.     

Immunocytochemical localization of L-glutamate decarboxylase, gamma-aminobutyric acid transaminase, cysteine sulfinic acid decarboxylase, aspartate aminotransferase and somatostatin in rat retina

The regional distribution and cellular location of GABA-synthesizing enzyme, L-glutamate decarboxylase (GAD), GABA degrading enzyme, GABA-transaminase (GABA-T), taurine synthesizing enzyme, cysteine sulfinic acid decarboxylase (CSAD), aspartate and glutamate converting enzyme, aspartate aminotransferase (AAT), and somatostatin have been visualized in the rat retina by immunocytochemical methods. GAD immunoreactivity was found to be concentrated in the inner plexiform layer. A moderate to weak staining of GAD was found in the inner nuclear layer. The distribution of GABA-T immunoreactivity was similar to that of GAD with the exception that a weak to moderate staining of GABA-T was also observed in the outer plexiform layer. CSAD immunoreactivity was seen in every layer with the heaviest staining in the inner plexiform layer, and moderate staining in the inner and outer nuclear layers and ganglion cell layer. AAT immunoreactivity was mostly concentrated in the outer nuclear layer; there was weak staining in the inner nuclear layer and inner and outer plexiform layer. Dense somatostatin staining was seen in the inner plexiform layer and moderate staining was present in the inner nuclear layer, outer plexiform layer and ganglion cell layer. These findings suggest that in rat retina, GABA-containing cells occur in some types of amacrine cells only, while taurine and somatostatin appear in both amacrine and horizontal cells. AAT immunoreactivity was primarily associated with the photoreceptor cells suggesting that AAT may be used as a marker for aspartergic/glutamergic cells and their endings in the central nervous system.     

Further pharmacological characterization of [3H]idazoxan binding sites in rat brain: evidence for predominant labeling of alpha 2-adrenergic receptors.

In addition to binding to alpha 2-adrenergic receptors, the antagonist [3H]idazoxan has been reported to bind to non-adrenergic sites in a number of tissues and species. In the present study, the pharmacological nature of [3H]idazoxan binding sites in rat brain slices has been examined using radioligand binding and autoradiographic techniques. In Na2KHPO4 buffer, four drugs with high affinity for alpha 2-adrenergic binding sites were potent inhibitors of [3H]idazoxan binding, with the rank order of potency being RX821002 greater than phentolamine greater than yohimbine greater than (-)epinephrine. Non-linear regression analysis resolved all competition curves into two components, with the high affinity site representing the majority of total [3H]idazoxan binding. In autoradiographic studies performed in Na2KHPO4 buffer, all alpha 2-selective ligands displaced greater than or equal to 75% of total [3H]idazoxan binding to most brain regions. These findings indicate that the major component of [3H]idazoxan binding was to sites that are alpha 2-adrenergic in nature. [3H]Idazoxan binding was also examined in glycylglycine buffer. In contrast to binding in Na2KHPO4 buffer, the proportion of low affinity sites was significantly increased in glycylglycine buffer. Autoradiographic studies confirmed these findings. These pharmacological data are consistent with our previously reported conclusions that, under appropriate assay conditions, [3H]idazoxan predominantly labels alpha 2-adrenergic binding sites in rat brain. These sites are widely distributed and have pharmacological characteristics consistent with those previously reported for alpha 2A-adrenergic receptors.     

Quantitative autoradiography of muscarinic and benzodiazepine receptors in the forebrain of the turtle, Pseudemys scripta

The distribution of muscarinic and benzodiazepine receptors was investigated in the turtle forebrain by the technique of in vitro receptor autoradiography. Muscarinic binding sites were labeled with 1 nM 3H-quinuclidinyl benzilate (3H-QNB), and benzodiazepine sites were demonstrated with the aid of 1 nM 3H-flunitrazepam (3H-FLU). Autoradiograms generated on 3H-Ultrofilm apposed to tissue slices revealed regionally specific distributions of muscarinic and benzodiazepine binding sites that are comparable with those for mammalian brain. Dense benzodiazepine binding was found in the anterior olfactory nucleus, the lateral and dorsal cortices, and the dorsal ventricular ridge (DVR), a structure with no clear mammalian homologue. Muscarinic binding sites were most dense in the striatum, accumbens, DVR, lateral geniculate, and the anterior olfactory nucleus. Cortical binding sites were studied in greater detail by quantitative analysis of autoradiograms generated by using emulsion-coated coverslips. Laminar gradients of binding were observed that were specific for each radioligand; 3H-QNB sites were most dense in the inner molecular layer in all cortical regions, whereas 3H-FLU binding was generally most concentrated in the outer molecular layer and was least dense through all layers in the dorsomedial cortex. Because pyramidal cells are arranged in register in turtle cortex, the laminar patterns of receptor binding may reflect different receptor density gradients along pyramidal cell dendrites.     

Immunocytochemical localization of l-glutamate decar☐ylase, gamma-aminobutyric acid transaminase, cysteine sulfinic acid decar☐ylase, aspartate aminotransferase and somatostatin in rat retina

The regional distribution and cellular location of GABA-synthesizing enzyme, l-glutamate decar☐ylase (GAD), GABA degradating enzyme, GABA-transaminase (GABA-T), taurine synthesizing enzyme, cysteinesulfinic acid decar☐ylase (CSAD), aspartate and glutamate converting enzyme, aspartate aminotrasferase (AAT), and somatostatin have been visualized in the rat retina by immunocytochemical methods. GAD immunoreactivity was found to be concentrated in the inner plexiform layer. A moderate to weak staining of GAD was found in the inner nuclear layer. The distribution of GABA-T immunoreactivity was similar to that of GAD with the exception that a weak to moderate staining of GABA-T was also observed in the outer plexiform layer. CSAD immunoreactivity was seen in every layer with the heaviest staining in the inner plexiform layer, and moderate staining in the inner and outer nuclear layers and ganglion cell layer. AAT immunoreactivity was mostly concentrated in the outer nuclear layer; there was weak staining in the inner nuclear layer and inner and outer plexiform layer. Dense somatostatin staining was seen in the inner plexiform layer and moderate staining was present in the inner nuclear layer, outer plexiform layer and ganglion cell layer. These findings suggest that in rat retina, GABA-containing cells occur in some types of amacrine cells only, while taurine and somatostatin appear in both amacrine and horizontal cells. AAT immunoreactivity was primarily associated with the photoreceptor cells suggesting that AAT may be used as a marker for aspartegic/glutamergic cells and their endings in the central nervous system.     

Binding properties, regional ontogeny and localization of adrenergic receptors in chick brain

The pharmacological properties of [3H]-WB4101, [3H]-clonidine and [3H]-dihydroalprenolol binding in chick brain membranes display the characteristics known for alpha 1-, alpha 2- and beta-adrenergic binding sites, respectively. Kinetic studies performed at different embryonic and post-hatching ages have shown one binding component for each one of the above radioactive ligands. The ontogeny of alpha 1, alpha 2 and beta binding sites was studied in cerebral hemispheres, optic lobes, brain stem and cerebellum. In all brain regions studied, the development of alpha 2 binding sites precedes that of alpha 1 and beta, and a very significant decrease of alpha 2 number was observed in the cerebellum, brain stem and optic lobes afterwards. The autoradiographic localization of adrenergic receptors was studied in the optic lobes and cerebellum. In the optic lobes the superficial layers of stratum griseum and fibrosum showed a strong selective labelling of alpha 1, alpha 2 and beta binding sites and the strong selective labelling of alpha 2 binding sites extended to the layer of stratum opticum. Among the nuclei located in the optic lobe only the nucleus mesencephalis lateralis pars dorsalis (MLD) exhibited a strong selective labelling for alpha 1 binding sites while, for beta binding sites, not only the MLD, but also the nucleus isthmic pars parvocellularis (Ipc) and the nucleus isthmic pars magnocellularisa (Imc) exhibited strong labelling. In the cerebellum strong selective labelling for alpha 1 and beta receptors was seen in the molecular layer. Labelling of the granule cell layer was almost equally strong for alpha 1 but significantly less for beta binding sites. No significant labelling could be detected for alpha 2 binding sites.     

3H]Spermine binding to synaptosomal membranes from the chick retina.

The binding of [3H]spermine to synaptosomal membranes from chick retina was examined. Saturable specific binding of [3H]spermine to synaptosomal membranes from plexiform layers of retina (P1 and P2) has been characterized, and found to concentrate in the inner plexiform layer compared to the outer plexiform layer (Bmax=9.3 and 37 pmol/mg protein for P1 and P2, respectively). Kinetics of specific [3H]spermine binding yield a sigmoidal saturation curve, indicating positive cooperativity (nH: 2.4 and 3.2 for P1 and P2, respectively) with high affinity: Kapp=61 and 67 nM for P1 and P2. The time required to attain equilibrium at room temperature was less than 5 min in both fractions. Dose-response curves for spermine, spermidine, and diethylene-triamine (DET) show different potencies for inhibiting [3HDET. Our results support a role for polyamines (PA) as neurotransmitters or neuromodulators in the vertebrate retina.     

[H]Spermine binding to synaptosomal membranes from the chick retina

The binding of [³H]spermine to synaptosomal membranes from chick retina was examined. Saturable specific binding of [³H]spermine to synaptosomal membranes from plexiform layers of retina (P₁ and P₂) has been characterized, and found to concentrate in the inner plexiform layer compared to the outer plexiform layer (B_max=9.3 and 37 pmol/mg protein for P₁ and P₂, respectively). Kinetics of specific [³H]spermine binding yield a sigmoidal saturation curve, indicating positive cooperativity (nH: 2.4 and 3.2 for P₁ and P₂, respectively) with high affinity: K_app=61 and 67 nM for P₁ and P₂. The time required to attain equilibrium at room temperature was less than 5 min in both fractions. Dose–response curves for spermine, spermidine, and diethylene–triamine (DET) show different potencies for inhibiting [³H]spermine binding: spermine>spermidine>DET. Our results support a role for polyamines (PA) as neurotransmitters or neuromodulators in the vertebrate retina.     

Receptor autoradiography in thoracic spinal cord: correlation of neurotransmitter binding sites with sympathoadrenal neurons

Receptor autoradiography was combined with the retrograde labeling of sympathoadrenal neurons by fast blue to determine whether opiate, serotonin, catecholamine, or cholinergic binding sites could be spatially correlated with preganglionic neurons in the rat intermediolateral cell column (IML) that project to the adrenal gland. [3H]Dihydromorphine (DHM) was used for the visualization of mu opiate receptors, [3H]lysergic acid diethylamide (LSD) for serotonin receptors, [3H]para-aminoclonidine (pAC) for alpha 2-adrenergic receptors, and [3H]quinuclidinyl benzilate (QNB) for muscarinic cholinergic receptors. While qualitative assessment of autoradiograms indicated that alpha 2-adrenergic and muscarinic receptors were concentrated in the intermediate zone of the spinal cord, quantitation of grains in specific regions of the intermediate gray revealed that alpha 2-adrenergic and serotonergic receptors were more highly concentrated over sympathoadrenal preganglionic neurons than over other regions in IML or the adjacent intermediate gray matter. Information concerning the distribution of neurotransmitter-binding sites in other regions of thoracic spinal cord was also obtained. All ligands showed relatively dense binding sites in the superficial laminae of the dorsal horn, and all but [3H]DHM revealed similar densities of binding sites in the region adjacent to the central canal. Only [3H]QNB revealed a high density of binding sites in the ventral horn of the spinal cord.     

Is taurine a neurotransmitter in rabbit retina?

Rabbit retina was used as a model to study the possible role of taurine in the retina. The taurine-synthesizing enzyme, cysteine sulfinic acid decarboxylase (CSAD), is localized immunohistochemically using specific antibodies against CSAD. The CSAD-immunoreactivity appears to be most prominent in the inner nuclear layer (INL) and ganglion cell layer (GCL). The inner plexiform layer (IPL), the outer nuclear layer and outer plexiform layer are sporadically stained. The CSAD-positive neurons include some amacrine cells and probably the bipolar cells in the INL and some large and small ganglion cells in the GCL. Autoradiographic studies reveal that the uptake of [3H]taurine is most prominent in the INL. The IPL and GCL, as well as the Müller cells, also show a moderate degree of [3H]taurine accumulation. In conclusion, we have demonstrated the presence of the taurine-synthesizing enzyme and uptake systems in rabbit retina. Based on the above evidence, we propose that taurine may be used by some neurons, presumably amacrine cells, as a transmitter in the rabbit retina.     

Heterogeneous binding of [3H]4-DAMP to muscarinic cholinergic sites in the rat brain: evidence from membrane binding and autoradiographic studies.

The present study shows that [3H]4-DAMP binds specifically, saturably, and with high affinity to muscarinic receptor sites in the rat brain. In homogenates of hippocampus, cerebral cortex, striatum, and thalamus, [3H]4-DAMP appears to bind two sub-populations of muscarinic sites: one class of high-affinity, low capacity sites (Kd less than 1 nM; Bmax = 45-152 fmol/mg protein) and a second class of lower-affinity, high capacity sites (Kd greater than 50 nM; Bmax = 263-929 fmol/mg protein). In cerebellar homogenates, the Bmax of [3H]4-DAMP binding sites was 20 +/- 2 and 141 +/- 21 fmol/mg protein for the high- and the lower-affinity site, respectively. The ligand selectivity profile for [3H]4-DAMP binding to its sites was similar for both the high- and lower-affinity sites; atropine = (-)QNB = 4-DAMP much greater than pirenzepine greater than AF-DX 116, although pirenzepine was more potent (16-fold) at the lower- than at the high-affinity sites. The autoradiographic distribution of [3H]4-DAMP sites revealed a discrete pattern of labeling in the rat brain, with the highest densities of [3H]4-DAMP sites present in the CA1 sub-field of Ammon's horn of the hippocampus, the dentate gyrus, the olfactory tubercle, the external plexiform layer of the olfactory bulb and layers I-II of the frontoparietal cortex. Although the distribution of [3H]pirenzepine sites was similar to that of [3H]4-DAMP sites in many brain regions, significant distinctions were apparent. Thus, both the ligand selectivity pattern of [3H]4-DAMP binding and the autoradiographic distribution of sites suggest that although the high-affinity [3H]4-DAMP sites may consist primarily of muscarinic-M3 receptors, the lower-affinity [3H]4-DAMP sites may be composed of a large proportion of muscarinic-M1 receptors.     

Neurotransmitter receptor localizations: Brain lesion induced alterations in benzodiazepine, GABA, β-adrenergic and histamine H1-receptor binding

Selective neuronal lesions have been utilized in efforts to localize binding sites in rat brain for β-adrenergic, γ-aminobutyric acid (GABA), histamine H₁ and benzodiazepine receptors. The various receptors respond differentially to lesions both in extent of change and in time course. After kainate lesions in the corpus striatum, benzodiazepine receptors are depleted up to 45% at 45–78 days but are unaffected after 7 days. By contrast striatal GABA receptors are increased at 7 days but depleted at later times. Thus both striatal benzodiazepine and GABA receptors appear to be associated at least in part with intrinsic neurons.In the cerebellum both benzodiazepine and GABA receptors are reduced in kainate treated rats and in Nervous mice, mutants which lack Purkinje cells. The most pronounced dissimilarity between benzodiazepine and GABA receptors occurs in Weaver mice, which selectively lack granule cells and display a 60% reduction in GABA receptors but a 40% augmentation in benzodiazepine receptors. A major portion of cerebellar GABA receptors, therefore, appear to be localized to granule cells.Striatal β-adrenergic receptors are reduced following intrastriatal kainate injections but are unaffected by cerebral cortex ablation, suggesting an association with intrinsic neurons but not with axon terminals of the corticostriate pathway. While intraventricular injections of 6-hydroxydopamine enhance [³H]dihydroalprenolol binding to β-adrenergic receptors in the cerebral cortex and hippocampus, such binding is not augmented in the corpus striatum, brain stem, midbrain or thalamus-hypothalamus by this treatment. Moreover, medial forebrain bundle lesions, which destroy ascending adrenergic neurons, fail to alter cerebral cortical or striatal β-adrenergic receptors. Thus denervation-elicited increase in β-adrenergic receptors vary with brain region and the type of denervating lesion.Histamine H₁-receptors are the most resistant of all to neuronal lesions. In the corpus striatum [³H]mepyramine binding is unaffected by cerebral cortex ablation, nigral injections of 6-hydroxydopamine or brain stem hemisection. In the hippocampus, medial forebrain bundle lesions, intrahippocampal kainate injection, and fimbria and fornix transection largely fail to alter [³H]mepyramine binding. Accordingly, a major portion of these receptors may be associated with nonneuronal elements such as glia or blood vessels.