Intrastriatal injection of [H]dopamine through a chronic cannula to produce rotation: Distribution and concentration of the tracer in specific brain regions

As others have shown, rats rotated contralaterally following unilateral intrastriatal injections of dopamine. However, the concentration of the injection solution necessary to elicit rotation in our and others' studies was high, 50 μg/0.5 μl (10,000 times endogenous concentration) and the latency to turn was 20–60 min. Thus, we injected [³H]dopamine ([³H]DA) to determine the concentration of the injected DA in the striatum and to determine if the long latency to turn was related to spread of DA over time to distal nuclei such as the globus pallidus, subthalamic nucleus and substantia nigra. Microinjections (0.5 μl) of [³H]DA were made through a chronic striatal cannula in pargyline-treated freely moving rats. Determinations of [³H]DA concentrations in individual brain areas were made at 20 min and 60 min following intrastriatal injection. The major accumulation of [³H]DA and its metabolites was in the dorsal striatum, near the cannula tip, and in the sensorimotor cortex where the cannula passed through to the striatum. The concentration of [³H]DA in the dorsal striatum was only 10–100 times the endogenous concentration, depending upon the cannula injection system used. The 60 min latency to rotate could not be attributed to spread of DA to the subthalamic n., entopeduncular n. and substantia nigra. Rotation following an intrastriatal injection of DA was dependent upon the dorsal striatal tissue concentration of DA. The ventral striatum and globus pallidus were also possibly involved. The highest striatal [³H]DA concentrations were, at most, 10 times greater than the concentrations of systemically injectedl-[³H]DOPA, and, at the lower concentrations which produced twisting and weak rotation, the DA concentration was similar to that seen with systemicl-DOPA.     

In Vivo release by vagal stimulation of L-[3H] glutamic acid in the nucleus tractus solitarius preloaded with L-[3H] glutamine

In anesthetized and paralyzed rats, using a push-pull perfusion technique, we examined the effect of bilateral vagal stimulation on the release of L-[³H] glutamic acid (L-[³H] Glu) from the nucleus tractus solitarius (NTS), after preloading the tissue either with L-[³H] Glu or L-[³H] glutamine (L-[³H] Gln). Vagal stimulation sufficient to produce a maximum fall of arterial pressure (AP) evoked release of L-[³H] Glu from the NTS when the tissue was preloaded with either ³H-Glu or ³H-Gln, and of D-[³H] aspartic acid (D-[³H) Asp) when this stable Glu analogue was used to preload the tissue. Results demonstrate that the precursor L-Gln is a good marker of the releasable pool of L-Glu in vivo and are consistent with the hypothesis that L-[³H] Glu is a neurotransmitter in the NTS, mediating the vasodepressor response from cardiopulmonary mechanoreceptors.     

Computer-assisted image analysis to quantify regional and specific receptor ligand binding: Upregulation of [3H]QNB and [3H]WB4101 binding in denervated hippocampus

Quantitative regional analysis of receptor autoradiographs using the Nikon Magiscan image analysis system permits resolution of regional variations in specific binding in non-homogeneous CNS structures, such as the hippocampus. Cholinergic denervation, produced by fimbrial transections, elicits a 24% increase in atropine-displaceable [³H]QNB binding in whole coronal sections of the hippocampal formation, which is greatest in the dorsal subiculum, CA3 and dentate gyrus. This lesion also elicits a 69% increase in lower affinity [³H]WB4101 binding which is displaceabte by phentolamine, but not by prazosin. This represents a sum of increases and decreases in binding in several subregions. Taken together, these findings serve to emphasize the need for normalized regional evaluation of subtracted images which have been calibrated, and linearized or transformed, to reveal binding specific to a single site.     

Opiate binding to brain slices and ontogenesis of hypothalamic [3H]naloxone binding sites

We have developed a radioligand binding assay based on the use of hypothalamic slices and have examined the ontogenesis of [³H]naloxone binding sites in male and female rats. [³H]NAL binding is reversible, saturable, stereospecific, of high affinity, readily displaceable by morphine and is sensitive to phenoxybenzamine. These characteristics suggest that [³H]NAL readily binds to opiate receptors in brain slices. With this assay we have demonstrated that: (a) there is an age-related increase in opiate binding sites in rat hypothalamus, (b) there are sex differences in the binding affinity of the sites and (c) the values of B_max are approximately 2–5-fold higher than the levels previously reported from assays with brain homogenates.     

Effects of NaCl and sultopride on striatal [3H]spiperone binding in neonatal,adult and senescent rats

Effects of NaCl, (+)-and (?)-sultopride on striatal [³H]spiperone binding was investigated in 7-day, 70-day and 2-year-old rats. The amount of specific [³H]spiperone binding was the highest at 70 days and the value at adult stage was significantly (P < 0.001) higher than those at 7 days and 2 years. NaCl (100 mM) significantly increased [³H]spiperone binding in neonatal (P < 0.01), adult (P < 0.05) and senescent (P < 0.05) animals. Scatchard analysis showed that the Bmax of low-affinity [³H]spiperone binding was significantly elevated by 100 mM NaCl compared to the value in control of adult animals. More potent inhibition of (?)-sultopride for [³H]spiperone binding than that of the (+)-enantiomer at adult stage was also observed at neonatal and senescent stages. NaCl (100 mM) significantly enhanced inhibitory activities of (+)- and (?)-sultopride at every stage. It is suggested that stabilizing effect of Na⁺ on dopamine (DA) receptor complexes and increasing effect of Na⁺ on binding affinity of benzamide to DA₂ receptors keep functions through development and aging.     

Distribution of serotonin and dopamine receptors in aplysia tissues: Analysis by [3H]LSD binding and adenylate cyclase stimulation

The distribution of receptors for serotonin and dopamine has been studied in various neuronal and non-neuronal tissues from Aplysia californica using: (1) a [³H]LSD binding assay; and (2) stimulation of adenylate cyclase activity. High levels of specific [³H]LSD binding were found in all ganglia and nerves examined. Lower levels of binding were present in a number of muscle tissues and in the sheath surrounding the central ganglia. The ability of serotonin and dopamine to inhibit [³H]LSD binding depended upon the tissue examined. In muscle tissue, most of the binding was sensitive to serotonin. In contrast, a number of ganglia (e.g. the pleural, abdominal or cerebral) contained a considerable proportion of dopamine-sensitive binding. A limited pharmacological analysis of serotonin-sensitive [³H]LSD binding indicated that Aplysia serotonin receptors are closely related to those found in the snail, Helix pomatia, and in rat brain. Adenylate cyclase activity in membranes from Aplysia ganglia, muscles and connective nerves was stimulated by serotonin (but not by dopamine). The amount of serotonin-sensitive adenylate cyclase correlated well with the amount of serotonin-sensitive [³H]LSD binding in most tissues.d-LSD was a partial agonist on the serotonin-sensitive adenylate cyclase, whereas the pharmacologically inactive stereoisomer l-LSD was without effect.The high density of serotonin receptors in pleuro-abdominal connective nerves, and their presence in the connective tissue sheaths surrounding the ganglia, suggests that not all of these receptors are located at synapses. On the other hand, the tissue distribution of dopamine and serotonin receptors, as measured by these techniques, is consistent with that expected from electrophysiological data.     

Stimulation increases incorporation of [3H]lysine into rat brain and liver proteins

Exposure of rats to electric footshocks or merely to the footshock apparatus increased the incorporation of [³H]lysine into brain and liver protein. The effect was present in both fore- and hindbrain. The footshock treatment was the more effective stimulus, producing larger and more significant changes. These results resemble those previously observed in C57B1/6J mice, and thus suggest that altered protein or amino acid metabolism is a general response to stress in rodents.     

Changes in pyruvate dehydrogenase complex (PDHc) activity and [3H]QNB-receptor binding in rat brain subsequent to intracerebroventricular injection of bromopyruvate

Summary Pyruvate dehydrogenase complex (PDHc), a link between carbohydrate and acetylcholine metabolism, is a regulatory enzyme for glucose and neurotransmitter metabolism in the brain and is reduced in Alzheimer-diseased brain. To study functional consequences of an inhibition of PDHc on muscarinic receptor binding, bromopyruvate, a suicide inhibitor of PDHc, was injected intracerebroventricularly (icv) in rats. Bromopyruvate caused a reduction of PDHc activity in the 3 brain regions examined, however, reaching significance only in the cerebral cortex and the hippocampus and not in the striatum, 24h after injection. 3, 6, and 12 weeks later, there was a normalization or transiently increased activity, respectively, of PDHc in these brain regions. No changes in concentrations of energy-rich phosphates could be demonstrated in the cerebral cortex 12 weeks after brompyruvate injection. The number of muscarinic receptors was significantly reduced in the cerebral cortex 12 weeks after injection. the data indicate that a transient reduction of brain PDHc activity in vivo is associated with a long-lasting reduction in muscarinic cholinergic receptors. Because comparable changes of PDHc and muscarinic receptors are found in dementia of Alzheimer type, the model of bromopyruvate inhibition of PDHc in rats is suggested to be useful for experimental dementia research.     

Effect of maternal nicotine on the development of sites for [3H]nicotine binding in the fetal brain

The sites for [³H]nicotine binding in fetal brains were examined after administration of nicotine into pregnant rats. Administration of unlabelled nicotine into the pregnant rats increased Bmax values for the sites for [³H]nicotine binding without affecting K_d values in the fetal brains. Treatment with this regimen, however, did not show any significant change in the sites for [³H]quinuclidinyl benzylate (QNB) binding. In addition, treatment with this regimen increased Bmax values of the sites for [³H]nicotine binding in the brains of pregnant rats. α-Bungarotoxin had no effect on the sites for [³H]nicotine binding.It is inferred, therefore, that a similar response is elicited by nicotine binding sites to administered nicotine in both the fetal and maternal brains. Furthermore, a possible effect of nicotine in pregnant rats may be the facilitation of the development of nicotine acetylcholine receptors in the fetal brain.     

Existence of [3H]serotonin binding sites in the rat spinal cord: A developmental study

[³H]5-HT specific binding sites have been characterized in the rat spinal cord. Experimental conditions allowed us to study a single class of sites possibly related to the postsynaptic receptor for 5-HT. Binding constants (K_D and B_max) are described for adult and developing animals. No substantial changes in affinity were observed but the number of receptors increased from birth up to day 20 postnatally, stabilizing thereafter. The developmental pattern of a presynaptic marker, tryptophan-5-hydroxylase, was similar to that of binding sites.     

[3H]Mepyramine binding sites in the optic lobe of the locust: autoradiographic and pharmacological studies

The presence and distribution of [³H]mepyramine binding sites was examined in the optic lobe of the locust in an attempt to identify the presence of histamine H1 receptors. Studies were carried out using both autoradiographic and ligand binding techniques. Although regions of the optic lobe showed significant binding, it appeared that in the insect nervous system [³H]mepyramine is not specific for the histamine H1 receptor.     

l-aspartate binding sites in rat cerebellum: A comparison of the binding ofl-[3H]aspartate andl-[3H]glutamate to synaptic membranes

The binding ofl-[³H]aspartate to sonicated, extensively washed and preincubated cerebellar synaptic membranes was investigated. Binding was optimal under physiological conditions of pH and temperature, and attained equilibrium within 10 min. Binding was saturable, and Eadie-Hofstee analysis revealed interaction with a single population of binding sites (K_d = 874nM and B_max = 44pmol/mg protein), which displayed no cooperativity (Hill coefficient approx.= 1). Specific [³H]aspartate was readily and reversibly displaced by unlabelledl-aspartate (thed-isomer being less than half as active) with a half-life of dissociation of 32 sec. Quisqualate, 4-fluoroglutamate and 2-amino-4-phosphonobutyrate, which are good displacers of [³H]glutamate binding, were only weakly active against the aspartate system. The excitatory amino acid antagonists,dl-α-aminoadipate,dl-α-aminosuberate and HA-966 were effective displacers, but the proposed aspartate receptor-preferring agonist, N-methyl-d-aspartate was inactive. Kainic acid exhibited negligible affinity for the aspartate binding site, in common with that for glutamate.While freezing or cold storage of membranes resulted in diminished [³H]-aspartate binding, lyophilization was not only able to confer substantial stability, but induced a marked increase in affinity of the binding site.Differential effects of various cations on [³H]aspartate binding were observed — monovalent cations reduced, while divalent cations enhancedl-[³H]aspartate binding.     

Decreased glucose utilization in discrete brain regions of rat in thioacetamide-induced hepatic encephalopathy as measured with [3H]-deoxyglucose

To evaluate the possible contribution of bioenergetic failure in the particular brain regions to the pathomechanism of hepatic encephalopathy (HE), local cerebral metabolic rate for glucose (LCMRglue) was evaluated from [3H]-deoxyglucose uptake in frontal, visual and auditory cortex, striatum, cerebellum and medulla oblongata of rats with acute HE induced with a hepatotoxin--thioacetamide (TAA). HE caused a decrease of LCMRglue in all the regions studied. The strongest decrease (about 65%) was noted in hippocampus and cerebral cortex--the two regions rich in glutamatergic neurons. The results indicate a possible link between decreased energy metabolism and impaired excitatory, glutamatergic neurotransmission--the two factors whose contribution to HE has so far been implicated separately.     

Axon reaction in hypoglossal and dorsal motor vagal neurons of adult rat: Incorporation of [3H]leucine

Pairs of adult rats received [³H]leucine (i.p., 5 μCi/g body weight) 0.25, 1, and 16 h before killing and zero (unoperated control animals) and 1 to 164 days after unilateral cervical vagotomy and hypoglossal neurotomy. Grain counts and morphometric measurements were made on axotomized and uninjured neurons in histoautoradiographs of the medullary nuclei. Axotomized hypoglossal neurons, which largely survive the injury, both enlarged and incorporated increased amounts of tritiated leucine at each labeling interval, 3 through 28 days postoperatively. In the vagal dorsal motor nucleus (DMN), axotomized cells, which frequently die after neurotomy, enlarged slightly through 28 days postoperatively, then atrophied; DMN neurons increased amino acid uptake for a shorter period (days 7 through 14) than hypoglossal neurons. This increase achieved statistical significance only when the labeling intervals were 0.25 or 1.0 h. Neurons of the DMN contralateral to vagotomy also enlarged. Axotomized DMN neurons did not sustain increased protein synthesis as long as their hypoglossal counterparts and seemed to fail to increase synthesis of structural protein with long half-lives (16-h labeling interval). The frequently necrobiotic response of axotomized DMN neurons may relate to these phenomena. From these and earlier results, we conclude that axon reaction appears to differ fundamentally in peripheral and central neurons. This difference may have significance for research on regeneration in the central nervous system.     

Binding of the [125I]3β-(Iodophenyl)tropan-2β-carboxylic acid isopropyl ester to the dopamine transporter at a physiologically relevant temperature: Mutually exclusive binding and different ionic requirements for various uptake blockers and substrates

The present study describes the binding of cocaine analog [¹²⁵I]3β-(iodophenyl)tropan-2β-carboxylic acid isopropyl ester ([¹²⁵I]RTI-121), a highly selective ligand for the dopamine transporter (DAT), to rat striatal synaptosomal membranes at 37°C. Saturation analysis of [¹²⁵I]RTI-121 binding revealed a single binding site with similar affinity for RTI-121 at both 50 and 134 mM NaCl. However, the density of binding sites was reduced at 134 mM NaCl. Various uptake blockers and substrates of the DAT monophasically inhibited the specific binding of [¹²⁵I]RTI-121. Increasing the NaCl concentration from 50 mM to 134 mM enhanced the affinity of the substrate dopamine and amphetamine for the DAT, without affecting that of the uptake blockers. At 134 mM NaCl, the copresence of GBR12935, BTCP, cocaine, amphetamine, or dopamine decreased the affinity of RTI-121 to the extent predicted by a model in which the binding of all compounds is mutually exclusive. This, along with a different NaCl sensitivity for blockers and substrates, suggests that the two categories of compounds recognize nonidentical but overlapping binding domains on the DAT. In contrast, the mutually exclusive binding with similar NaCl sensitivity for RTI-121 and the other uptake blockers tested here suggests the involvement of common binding domains in the recognition of these blockers. Synapse 25:155–162, 1997. © 1997 Wiley-Liss, Inc.     

Muscarinic receptors in the central nervous system of the rat. II. Distribution of binding of [3H]propylbenzilylcholine mustard in the midbrain and hindbrain

The distribution of muscarinic receptors has been studied in the rat midbrain and hindbrain by counting silver grains in light microscope autoradiographs of the specific (atropine-sensitive) binding of [³H]propylbenztlylcholine mustard in cryostat sections. Of the 78 areas studied 6 had grain counts between 6 and 9 times the nonspecific level (‘high’), and a further 15 had counts 4–6 times non-specific (‘intermediate’).The basilar pontine nuclei and the ventral nuclei of the lateral lemniscus had high counts. Among the cranial nerve motor nuclei the facial and hypoglossal nuclei had high counts and the motor trigeminal nucleus and nucleus ambiguus had medium counts. The interpeduncular nucleus as a whole had low counts but there were two bands of intense staining on each side around the entry zone of the bundles of afferent cholinergic fibres from the habenula. Intermediate levels of binding occurred over the inferior colliculus and the superficial and intermediate grey layers of the superior colliculus. The molecular layer of the vestibulocerebellar vermis was distinctly labelled.     

Evidence for a Short Feedback of Prolactin on the Tubero-Infundibular Endings: Differential Effect on the Release of [3 H]Gamma-Aminobutyric Acid and [3 H]Dopamine from Superfused Median Eminence

Release experiments were undertaken on the tubero-infundibular terminals of the hypothalamic median eminence. Isolated median eminences were preloaded with [³ H]y-aminobutyric acid (GABA) or [³ H]dopamine in such conditions that non-specific uptake was pharmacologically impaired. Each median eminence was continuously superfused with Krebs bicarbonate medium and tritium releases were evoked with high potassium solutions. The evoked release was estimated by measuring the total radioactivity of which actual dopamine and GABA represented more than 60% and 80%, respectively. The experiments were done by applying two successive 5-min pulses of potassium, the second in the presence of prolactin. These stimulations were submaximal: for each, a KCl concentration was chosen that gave about half the maximal response (45 mM for GABA and 30 mM for dopamine); the responses were suppressed by calcium-free medium. Rat prolactin induced a significant increase of dopamine-evoked release in a dose-dependent manner with a maximum (30%) at 25 μg prolactin/ml of medium. Over the same range of concentration a significant but still weak (10%) increase of evoked GABA was observed only with the highest prolactin concentration (25 μg prolactin/ml). At the same concentrations, ovine prolactin induced a markedly reduced effect on dopamine release and had no effect on GABA release. Spontaneous releases of dopamine and GABA were not affected by prolactin. These results show that prolactin enhances acutely and directly, but differently, the evoked release of GABA and dopamine by the median eminence. This differential effect may reflect either a differential regulation or a greater heterogeneity of GABAergic endings. Our data give support for a short feedback exerted by prolactin directly on the tubero-infundibular neurons.