Properties of [3H](3-Me-His2)TRH binding to apparent TRH receptors in the sheep central nervous system

[3H](3-methyl-His2)thyrotropin releasing hormone ([3H]MeTRH) binds to sites in the sheep central nervous system (CNS) whose properties closely resemble both those of CNS binding sites for [3H]TRH and those of pituitary binding sites for [3H]MeTRH. Detailed studies for binding of [3H]MeTRH in the sheep nucleus accumbens and retina have yielded equilibrium dissociation constants of about 4 nM and densities of binding sites of about 3 and 2 pmol/g wet weight, respectively. The binding affinity of [3H]MeTRH was 8- to 10-fold higher than that of [3H]TRH, resulting in much lower non-specific binding with the new ligand. The association reaction had a rate constant of about 2-3 x 10(7) M-1 min-1, while the biphasic dissociation reaction had rate constants of 8-9 x 10(-2) min-1 for the fast phase and 1-2 x 10(-2) min-1 for the slow phase. The regional distribution of binding in the sheep CNS was similar to that observed previously with [3H]TRH. Highest binding outside the pituitary was in the nucleus accumbens area and retina, with another peak in the amygdala-temporal cortex area. Binding was widely distributed, so that no CNS region appeared totally devoid of binding. Nineteen TRH analogs, ranging in potency over 6 orders of magnitude, showed nearly identical abilities to complete for binding of [3H]MeTRH in the CNS areas and in the sheep anterior pituitary gland in side-by-side experiments. These findings argue strongly for identification of [3H]MeTRH binding sites in the CNS as TRH receptors.     

Thyrotropin-releasing hormone (TRH) receptors. Localization by light microscopic autoradiography in rat brain using [3H][3-Me-His2]TRH as the radioligand

Thyrotropin releasing hormone (TRH) is a putative neurotransmitter in both the central and peripheral nervous system. In the present report, we have used autoradiography coupled with densitometric analysis of tritium-sensitive film to investigate the distribution of [3H][3-Me-His2]TRH [( 3H]MeTRH)-binding sizes in the rat brain. Previous pharmacological reports have established that many of these [3H]MeTRH-binding sites have a structure-activity profile consistent with being a physiological TRH receptor. A high level of TRH receptors were observed in the accessory olfactory bulb, lateral nucleus of the amygdala, dentate gyrus, and entorhinal cortex. Moderate levels of TRH receptors were observed in the rhinal cortex, hypothalamus, superior colliculus, several brainstem motor nuclei, and lamina I of the spinal trigeminal nucleus pars candalis, while low concentrations of receptors are present in the cerebral cortex, striatum and ventral horn of the spinal cord. Very low levels of receptors were observed in the globus pallidus and in most nuclei of the dorsal thalamus. Comparisons of the distribution of TRH receptors to TRH-immunoreactive content indicates that, while in some areas of the brain there is a rough correlation between levels of TRH peptide and its receptor, in most brain areas there is little obvious correlation between the two. While such a discrepancy has been observed for other peptides and their receptors, the extensive distribution of TRH receptors in the central nervous system does provide an explanation for the variety of behavioral effects observed when TRH is infused into the central nervous system.     

Properties of [H](3-Me-His)TRH binding to apparent TRH receptors in the sheep central nervous system

[³H](3-methyl-His²)thyrotropin releasing hromone ([³H]MeTRH) binds to sites in the sheep central nervous system (CNS) whose properties closely resemble both those of CNS binding sites for [³H]TRH and those of pituitary binding sites for [³H]MeTRH. Detailed studies of binding of [³H]MeTRH in the sheep nucleus accumbens and retina have yielded equilibrium dissociation constants of about 4 nM and densities of binding sites of about 8 and 2 pmolf/g wet weight, respectively. The binding affinity of [³H]MeTRH was 8- to 10-fold higher than that of [³H]TRH, resulting in much lower non-specific binding with the new ligand. The association reaction had a rate constant of about 2–3 × 10⁷ M⁻¹ min⁻¹, while the biphasic dissociation reaction had rate constants of 8–9 × 10⁻² min⁻¹ for the fast phase and 1–2 × 10⁻² min⁻¹ for the slow phase. The regional distribution of binding in the sheep CNS was similar to that observed previously with [³H]TRH. Highest binding outside the pituitary was in the nucleus accumbens area and retina, with another peak in the amygdala-temporal cortex area. Binding was widely distributed, so that no CNS region appeared totally devoid of binding. Nineteen TRH analogs, ranging in potency over 6 orders of magnitude, showed nearly identical abilities to compete for binding of [³H]MeTRH in the CNS areas and in the sheep anterior pituitary gland in side-by-side experiments. These findings argue strongly for identification of [3H]MeTRH binding sites in the CNS as TRH receptors.     

Autoradiographic localization of alpha5 subunit-containing GABAA receptors in rat brain

Multiple subtypes of GABAA receptors are expressed in the rat central nervous system (CNS). To determine the distribution and proportion of alpha5 subunit containing receptors, quantitative autoradiographic analyses were performed with both [3H]L-655,708 and [3H]Ro15-1788, an alpha5 selective and a non selective benzodiazepine binding site ligand, respectively. High densities of [3H]L-655,708 binding sites were observed in hippocampus and olfactory bulb, where alpha5 receptors accounted for 20-35% of total [3H]Ro15-1788 binding sites. Low levels of [3H]L-655,708 sites were associated with the cortex as well as amygdala, thalamic, hypothalamic and midbrain nuclei. These observations indicate that although [3H]L-655,708 binding sites have an overall low expression in rat CNS, they may contribute significantly to GABAergic inhibition in specific brain regions.     

Autoradiographic distribution of TRH binding sites in the human hypothalamus.

Using in vitro quantitative autoradiography and [3H]3MeTRH, a selective high affinity radioligand, we examined the rostrocaudal distribution of TRH binding sites in both the infant and the adult human hypothalamus. The saturation curve shows that the [3H]3MeTRH binds with high affinity to a single class of TRH binding sites and is saturable, the apparent constant of dissociation is in the namomolar range. TRH binding sites showed a wide distribution, principally in the anterior and mediobasal levels of the hypothalamus. TRH binding site concentration was highest within the diagonal band of Broca, the lateral preoptic area, the infundibular and the tuberal nuclei. TRH binding site concentration was moderate in the ventromedial nucleus and the medial preoptic area, whereas we observed low densities in the periventricular, paraventricular and mammillary nuclei. The distribution in the infant and the adult is generally similar. However, it is noteworthy that the infant tuberal nuclei displayed a lower binding site density when compared to the adult. On the other hand, the diagonal band of Broca is relatively more labeled in infant. The analysis of the whole hypothalamus allows us to ascertain the absence of lateral asymmetric distribution both in the infant and the adult. No significant difference is noticed when considering as parameters of variation age, sex or post mortem delay.     

Receptors for thyrotropin-releasing hormone (TRH) in rabbit spinal cord

Specific binding of³H-labeled[3-Me-His²]TRH([³H]MeTRH) to membranes of rabbit spinal cord (thoracic) involved a homogeneous population of high-affinity sites (K_d = 2.7 ± 0.17 (n= 5)nM, B_max= 204 – 12(5)fmol/mg protein). TRH analogs competed for the binding in the following rank order of potency: MeTRH>TRH>TRH-Gly-NH₂ >Ser-His-Pro-NH₂ >Thr-His-Pro-NH₂ > pGlu-His-Pro-NH-C₂H₅>TRH-free acid. Competition experiments with rat amygdala, run in parallel with rabbit spinal cord, revealed a closely similar pattern of activity. These properties help identify binding sites in the rabbit spinal cord as physiological receptors for TRH. The binding sites resemble receptors previously demonstrated in pituitary and CNS tissues of other species.

The densities of [³H]MeTRH binding sites in different segments were generally similar, although density in the thoracic segment appeared to be somewhat higher. In all segments, binding seemed to be enriched in the dorsal gray matter. Dorsal roots and their associated ganglia, however, displayed little or no binding.     

The anticonvulsive action of adenosine: a postsynaptic, dendritic action by a possible endogenous anticonvulsant

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) is a potent neuroexcitatory compound which acts at the quisqualate class of excitatory amino acid receptors. In this study we describe the pharmacological characteristics and anatomical distribution of [3H]AMPA binding sites in rat brain using quantitative autoradiography. These binding sites exhibit the appropriate pharmacological characteristics and are found in high concentrations in the hippocampus, cerebral cortex (especially layers I-III), induseum griseum, and dorsal lateral septum. Intermediate concentrations are found in the corpus striatum and deeper layers of cerebral cortex. Lower concentrations are found in the diencephalon, midbrain and brainstem. These results demonstrate that [3H]AMPA binding sites are found throughout the CNS and suggest brain regions which may use quisqualate receptors as glutamate neurotransmitter receptors.     

Autoradiographic localization of α5 subunit-containing GABAA receptors in rat brain

Multiple subtypes of GABA_A receptors are expressed in the rat central nervous system (CNS). To determine the distribution and proportion of α5 subunit containing receptors, quantitative autoradiographic analyses were performed with both [³H]L-655,708 and [³H]Ro15-1788, an α5 selective and a non selective benzodiazepine binding site ligand, respectively. High densities of [³H]L-655,708 binding sites were observed in hippocampus and olfactory bulb, where α5 receptors accounted for 20–35% of total [³H]Ro15-1788 binding sites. Low levels of [³H]L-655,708 sites were associated with the cortex as well as amygdala, thalamic, hypothalamic and midbrain nuclei. These observations indicate that although [³H]L-655,708 binding sites have an overall low expression in rat CNS, they may contribute significantly to GABAergic inhibition in specific brain regions.     

Distribution of [H]AMPA binding sites in rat brain as determined by quantitative autoradiography

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) is a potent neuroexcitatory compound which acts at the quisqualate class of excitatory amino acid receptors. In this study we describe the pharmacological characteristics and anatomical distribution of [³H]AMPA binding sites in rat brain using quantitative autoradiography. These binding sites exhibit the appropriate pharmacological characteristics and are found in high concentrations in the hippocampus, cerebral cortex (especially layers I–III), induseum griseum, and dorsal lateral septum. Intermediate concentrations are found in the corpus striatum and deeper layers of cerebral cortex. Lower concentrations are found in the diencephalon, midbrain and brainstem. These results demonstrate that [³H]AMPA binding sites are found throughout the CNS and suggest brain regions which may use quisqualate receptors as glutamate neurotransmitter receptors.     

3H]Resiniferatoxin autoradiography in the CNS of wild-type and TRPV1 null mice defines TRPV1 (VR-1) protein distribution

Knowledge of the distribution and function of the vanilloid receptor (VR-1 or TRPV1) in the CNS lacks the detailed appreciation of its role in the peripheral nervous system. The radiolabelled vanilloid agonist [3H]resiniferatoxin (RTX) has been used to indicate the presence of TRPV1 receptor protein in the brain but low specific binding has complicated interpretation of this data. Recently, support for a more widespread CNS distribution of TRPV1 mRNA and protein has been provided by RT-PCR and antibody data. We have exploited the availability of TRPV1 null mice and used [3H]RTX autoradiography in the CNS of TRPV1 wild-type and TRPV1 null mice to identify the component of [3H]RTX binding to TRPV1 receptor protein. In the brains of TRPV1+/+ mice, specific [3H]RTX binding was broadly localised with the greatest binding in the olfactory nuclei, the cerebral cortex, dentate gyrus, thalamus, hypothalamus, periaqueductal grey, superior colliculus, locus coeruleus and cerebellar cortex. Specific binding was also seen in the spinal cord and sensory (dorsal root and trigeminal) ganglia. This binding was much lower but not abolished in most regions in the TRPV1-/- mice. Nonspecific binding was low in all cases. The present study unequivocally demonstrates a widespread and discrete distribution pattern of the TRPV1 receptor protein in the rat central nervous system. The presence of TRPV1 receptors in several brain regions suggests that it may function as a cannabinoid-gated channel in the CNS.     

Characterization and autoradiographic localization of TRH receptors in sections of rat brain

Receptors for thyrotropin-releasing hormone (TRH) in rat brain have been localized autoradiographically by exposing tritium-sensitive film to sections labeled with [3H]3-Me-His2-TRH. Greatest grain density was found over certain nuclei of the amygdala, with considerable density over several other forebrain areas. Properties of TRH receptor binding in frozen sections closely resembled those of receptors in fresh membrane fragments.     

Quantitative autoradiography of M2 muscarinic receptors in the rat brain identified by using a selective radioligand [3H]AF-DX 116

Muscarinic receptors of the M2 type have been studied in the rat brain using quantitative autoradiography with the selective ligand, [3H]AF-DX 116. High specific binding of [3H]AF-DX 116 was found in areas such as laminae IV and V of the parietal cerebral cortex, thalamus and hypothalamus and dentate gyrus. Intermediate [3H]AF-DX 116 binding was found in the frontal cortex, hippocampus, caudate-putamen, nucleus accumbens, and claustrum as well as in certain brainstem nuclei such as the nucleus of the solitary tract and the dorsal motor nuclei of the vagus. In contrast, the accessory olfactory nucleus, globus pallidus and cerebellum contained very low concentrations of M2 receptors. The present study demonstrates a unique regional distribution of M2 receptors in the rat brain.     

Quantitative autoradiographic distribution of [3H]AF-DX 116 muscarinic-M2 receptor binding sites in rat brain

The distribution of muscarinic-M2 receptors in rat brain was investigated by in vitro autoradiography using [3H]AF-DX 116, a putative probe for the muscarinic-M2 receptor subtype. Incubation of rat brain coronal sections with 10 nM [3H]AF-DX 116 showed highest binding site densities in discrete areas such as the superior colliculus and certain thalamic and brainstem nuclei, similar to the distribution reported for [2H]acetylcholine/M2 sites. [3H]AF-DX 116 site densities were markedly lower in forebrain areas such as cortex, striatum, and hippocampus, in contrast to the distribution seen for [3H]pirenzepine-M1 binding sites, which were concentrated in these forebrain areas; however, differential patterns of labeling were observed for the two muscarinic-M2 probes, [3H]AF-DX 116 and [3H]acetylcholine, in the hippocampal formation. Although [3H]AF-DX 116 binding was broadly distributed in multiple subfields of the hippocampus, [3H]acetylcholine binding was discretely distributed in a manner resembling that of acetylcholinesterase staining. This suggests the existence of muscarinic-M2 subtypes in the CNS, especially in the hippocampal formation.     

Quantitative autoradiography of [3H]nitroimipramine binding sites in rat brain

We have used the recent tritium-sensitive film method of quantitative autoradiography to localize in rat brain high-affinity binding sites for nitroimipramine (NI), a long-lasting inhibitor of serotonin (5-HT) transport in platelets. The distribution of NI binding sites in rat brain closely parallels the location of 5-HT terminals. There are high concentrations of binding sites in the dorsal and medial raphe nuclei, the basal portion of the mammilary nuclei, the medial forebrain bundle, the olfactory tubercule and the posterior basal nucleus of the amygdala. The association of [3H]NI binding sites with regions having a high content of 5-HT supports the notion that the high-affinity binding site for [3H]NI corresponds to some aspect of the presynaptic uptake site for 5-HT.     

Seasonal and state-dependent changes in brain TRH receptors in hibernating ground squirrels.

Quantitative autoradiography was used to localize and quantify thyrotropin-releasing hormone (TRH) receptors in the brain of hibernating (H), winter euthermic (WE), and summer euthermic (SE) animals to further explore the state-dependent physiological and behavioral effects of TRH in ground squirrels. The pattern of [3H]MeTRH binding (Kd 6.7 +/- 0.1 nM) was heterogeneous and highly concentrated in structures primarily associated with the limbic forebrain. Statistically significant seasonal changes (SE vs. WE) were reflected by decreases in TRH receptor binding in the arcuate nucleus, dorsomedial nucleus, and ventral pallidum of WE animals. Increased binding in WE animals was evident in the suprachiasmatic nucleus and choroid plexus of the lateral ventricles. Significant state-dependent changes (WE vs. H) were characterized by decreases in TRH receptor binding in the hypothalamic paraventricular nucleus, medial preoptic area, ventral tegmental area, and choroid plexus of the lateral ventricles of H animals. Increased binding occurred in the anterior cortical nucleus of the amygdala in H animals. The results suggest that naturally occurring changes in central TRH systems may be important in the mediation of physiological and behavioral processes that undergo seasonal and state-dependent adjustments in hibernating mammals.     

Quantitative autoradiography of [H]nitroimipramine binding sites in rat brain

We have used the recent tritium-sensitive film method of quantitative autoradiography to localize in rat brain high-affinity binding sites for nitroimipramine (NI), a long-lasting inhibitor of serotonin (5-HT) transport in platlets. The distribution of NI binding sites in rat brain closely parallels the location of 5-HT terminals. There are high concentrations of binding sites in the dorsal and medial raphe nuclei, the basal portion of the mammilary nuclei, the medial forebrain bundle, the olfactory tubercule and the posterior basal nucleus of the amygdala. The association of [³H]NI binding sites with regions having a high content of 5-HT supports the notion that the high-affinity binding site for [³H]NI corresponding to some aspect of the presynaptic uptake site for 5-HT.     

A topographic quantitative method for measuring brain tissue pH under physiological and pathophysiological conditions

The autoradiographic regional localization of [3H]beta-endorphin binding in rat brain differed from that of either [3H]dihydromorphine or [3H]D-Ala2-D-Leu5-enkephalin. Comparisons were made from sequential sections through 3 regions of rat brain: striatum, hypothalamus/thalamus, and brainstem. [3H]beta-endorphin labeled some clusters as well as the subcallosal streak in the striatum, the nucleus accumbens, lamina IV of the cortex, medial regions of the thalamus, hippocampus, inferior colliculus, dorsal raphe, median raphe and pontine nuclei. White matter regions had little binding. Although many of these structures were also labeled with either [3H]dihydromorphine or [3H]D-Ala2-D-Leu5-enkephalin, the overall pattern of [3H]beta-endorphin labeling appeared unique, consistent with the proposal of central epsilon receptors.     

AMPA receptor potentiation by acetylcholinesterase is age-dependently upregulated at synaptogenesis sites of the rat brain

We have used radioligand binding to synaptic membranes from distinct rat brain regions and quantitative autoradiography to investigate the postnatal evolution of acetylcholinesterase (AChE)-evoked up-regulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors in CNS areas undergoing synaptogenesis. Incubation of synaptosomal membranes or brain sections with purified AChE caused a developmentally modulated enhancement in the binding of [3H]-(S)-AMPA and the specific AMPA receptor ligand [3H]-(S)-5-fluorowillardiine, but did not modify binding to kainate neither N-methyl-D-aspartate receptors. In all postnatal ages investigated (4, 7, 14, 20, 27, 40 days-old and adult rats), AChE effect on binding was concentration-dependent and blocked by propidium, BW 284c51, diisopropylfluorophosphonate and eserine, therefore requiring indemnity of both peripheral and active sites of the enzyme. AChE-mediated enhancement of [3H]-fluorowillardiine binding was measurable in all major CNS areas, but displayed remarkable anatomical selectivity and developmental regulation. Autoradiograph densitometry exhibited distinct temporal profiles and peaks of treated/control binding ratios for different cortices, cortical layers, and nuclei. Within the parietal, occipital and temporal neocortices, hippocampal CA1 field and cerebellum, AChE-potentiated binding ratios peaked in chronological correspondence with synaptogenesis periods of the respective AMPA-receptor containing targets. This modulation of AMPA receptors by AChE is a molecular mechanism able to transduce localized neural activity into durable modifications of synaptic molecular structure and function. It might also contribute to AChE-mediated neurotoxicity, as postulated in Alzheimer's disease and other CNS disorders.     

Distribution and binding parameters of GABA and benzodiazepine receptors in the cat motor thalamus and adjacent nuclear groups

Quantitative receptor binding autoradiography technique was utilized to study GABA and benzodiazepine receptors in the cat motor thalamus (ventral anterior, ventral medial and ventral lateral nuclei) and adjacent thalamic subdivisions. Binding parameters (Bmax and Kd) and distribution pattern of the binding sites for 3 tritiated ligands [3H]muscimol ([3H]MUS), [3H]flunitrazepam ([3H]FLU) and [3H]baclofen ([3H]BAC) were analyzed and compared using measurements from discrete and anatomically well-defined thalamic regions. There was little correlation in the regional distribution of the 3 binding sites. The concentration of [3H]BAC binding sites in thalamic nuclei of interest was very low, practically at the limit of resolution of the quantitative autoradiographic technique; whereas appreciable quantities of [3H]MUS and [3H]FLU binding sites were present in the motor and adjacent limbic nuclei of the thalamus. There was more difference between the nuclei in regard to the number of high affinity GABA receptors than benzodiazepine receptors. Moreover, the ratio of Bmax[3H]MUS/Bmax[3H]FLU varied from 2.2 to 4.4 in different thalamic regions suggesting the presence of a diverse population of GABAA and benzodiazepine receptors. The distribution pattern of the 3 binding sites was compared to the topography of GABAergic afferents of the basal ganglia origin and the frequency of GABAergic synapses formed by thalamic local circuit neurons (LCN) in the motor thalamus that were established earlier. It was concluded that in the cat motor thalamus: (1) none of the ligands studied appear to reveal the receptors associated with nigro- or pallidothalamic synapses; (2) [3H]MUS binding sites may be associated with the dendrodendritic contacts formed by LCN; and (3) the [3H]FLU binding sites are physically unrelated to [3H]MUS binding sites. The concentration of [3H]FLU and [3H]MUS binding sites in the midline nuclei and of [3H]MUS binding sites in the limbic nuclei was remarkably high. It was concluded that in addition to previously suggested limbic structures, the midline nuclei with their very high content of benzodiazepine receptors may be considered as a neuroanatomical substrate of certain forms of anxiety.     

Ontogenesis of adenosine receptors in the central nervous system of the rat

The ontogeny of adenosine receptors was studied in rat brain and spinal cord using the specific ligand [3H]cyclohexyladenosine [( 3H]CHA). The [3H]CHA affinity constant (Kd) and the maximum receptor binding capacity (Bmax) were analyzed at all ages and in all CNS regions studied. Throughout development the Kd of [3H]CHA binding remained relatively stable and for cortex, cerebellum, subcortex, midbrain, brainstem and spinal cord ranged from 2.2 +/- 0.2 to 5.5 +/- 0.6 nM (mean +/- S.E.M.). In contrast, the Bmax values from 1- and 90-day animals increased by as little as 2-fold in subcortical regions and by as much as 9- and 16-fold in cortex and cerebellum, respectively. The highest density of binding sites was observed in subcortical structures and the lowest in brainstem and midbrain. In cortex, a steady increase in receptor number began at day 1 and stopped at the adult level by 21 days. In cerebellum, maximum receptor proliferation began at about 14 days and continued to adulthood. Other CNS regions showed intermediate rates of receptor development. These differences may reflect both the pattern of postnatal neurogenesis in the rat CNS and the maturation of those neural elements containing adenosine receptors.