Changes in cholinergic markers following kainic acid lesion of the ventral globus pallidus in rat

Choline acetyltransferase (CAT) and glutamate decarboxylase (GAD) activities and [3H] quinuclidinyl benzilate ([3H]QNB) binding were determined in the rat frontal cortex following damage to the basal forebrain cholinergic system. Pre- and postsynaptic changes in the cholinergic system with the passage of time were also studied. After a unilateral injection of kainic acid into the right ventral globus pallidus, the GAD levels remained unaffected, but the CAT levels decreased to 63.4% after 7 days. After 12 weeks, the CAT levels had returned to 87% of the control value. The Bmax of [3H]QNB binding for the muscarinic receptor was higher in the ipsilateral cortex up to 4 weeks. On the other hand, the KD value at 12 weeks was higher without a change in the Bmax of the [3H]QNB binding. These findings might indicate an ongoing compensatory receptor mechanism of denervation supersensitivity as a response to early changes in presynaptic cholinergic activity and the production of postsynaptic effect with presynaptic cholinergic damage over a long period of time.     

Effect of Forced-Running Stress on β-Adrenergic Receptors in Rat Brain Regions and Liver

Abstract: Rats were exposed to forced-running stress for 1 day, 3 days or a long term (approximately 2 weeks), and P-adrenergic receptor binding was then assayed using [³H]dihydroalprenoloI (DHA) in six brain regions and the liver. In the pons + med.obl., hypothalamus and midbrain, a reduction in Fadrenergic receptor density was first evident on day 1. In contrast, a decrease in Fadrenergic receptor density in the cerebral cortex and hippocampus was first evident on day 3. Decreased [³H]DHA binding in the pons + med.obl., cerebral cortex and hippocampus subsequently plateaued for the duration of the forced-running stress. In the midbrain and hypothalamus, however, decreased [³H]DHA binding subsequently returned to control levels despite the exposure to the forced-running stress. In the cerebellum and the liver, [³H]DHA binding did not change significantly throughout the stress. These results indicate that the forced-running stress induces both the time- and region-specific changes in Fadrenergic receptors. Moreover, the rats showed either a behavioral depression or a spontaneous recovery of running activity during the 2 weeks following the end of the long-term stress. Thus, we also examined the relationship of P-adrenergic receptors to these behavioral differences. [³H]DHA binding for the behavioral depression group was lower in the hippocampus and higher in the liver than for the spontaneous recovery group.     

Postischemic changes of [3H]nimodipine and [3H]ryanodine binding in the gerbil striatum and hippocampus

Sequential alterations of [³H]nimodipine and [³H]ryanodine binding in gerbils were investigated in selectively vulnerable regions, such as the striatum and hippocampus, 1 h to 7 days after 10 min of transient cerebral ischemia. [³H]Nimodipine binding showed no significant changes in the striatum and hippocampus up to 48 h after ischemia. Seven days after ischemia, however, a severe reduction in [³H]nimodipine binding was observed in the dorsolateral striatum, hippocampal CA1 (stratum oriens, stratum pyramidale and stratum radiatum) and hippocampal CA3 sector. On the other hand, [³H]ryanodine binding showed a significant increase in the hippocampus 1 h after ischemia. Five hours after ischemia, a significant reduction in [³H]ryanodine binding was observed only in the hippocampal CA1 sector. Thereafter, the striatum and hippocampus showed no significant alterations in [³H]ryanodine binding up to 48 h after ischemia. After 7 days, a marked reduction in [³H]ryanodine binding was observed in the striatum and hippocampus which were particularly vulnerable to ischemia. These results demonstrate that postischemic alteration in [³H]nimodipine and [³H]ryanodine binding is produced with different processes in the hippocampus. They also suggest that the mechanism for striatal cell damage caused by transient cerebral ischemia may, at least in part, differ from that for hippocampal neuronal damage. Furthermore, our findings suggest that abnormal calcium release from intracellular stores may play a pivotal role in the development of hippocampal neuronal damage.     

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.     

Release of Acetylcholine and Noradrenaline from the Cholinergic and Adrenergic Afferents in Rat Hippocampal CA1, CA3 and Dentate Gyrus Regions

An attempt was made to study the release of acetylcholine (ACh) and noradrenaline and their presynaptic modulation in isolated slice preparations dissected from different subfields of the hippocampus: CA1, CA3 and the dentate gyrus. The slices were perfused and loaded with [³H]choline or with [³H]noradrenaline. The release in response to field stimulation was determined radiochemically and the content of transmitters was assayed by a chemiluminescent method or by HPLC combined with electrochemical detection. After 30 min of loading with [³H]choline there were marked subregional differences in the specific activity of [³H]ACh content. The highest concentration was measured in the dentate gyrus and the lowest in CA3. Evidence was obtained that in all three subfields the cholinergic axon terminals are equipped with inhibitory muscarinic autoreceptors and the noradrenergic terminals with α₂-autoreceptors, as indicated by an increase in transmitter release when the tissue was exposed to selective muscarinic or α₂-adrenoceptor antagonists. In contrast, the cholinergic boutons are not equipped with α₂-adrenoceptors, and noradrenergic terminals do not possess inhibitory muscarinic receptors. It is therefore concluded that while the release of both ACh and noradrenaline is controlled by negative feedback modulation, there is no possibility of establishing a presynaptic inhibitory interaction between the two.     

Evidence For Two Neurochemical Divisions in the Human Nucleus Accumbens

The neurochemical anatomy of the human nucleus accumbens was studied by comparing the distributional patterns of [³H]DAMGE (μ opioid receptor), [³H]bremazocine (κ opioid receptor), [³H]SCH-23390 (D₁-like dopamine receptor), [³H]7-OH-DPAT (D₃ dopamine receptor) binding, preproenkephalin mRNA and acetylcholinesterase activity in sections of post mortem human striatum. Our results demonstrate the presence of at least two neurochemically distinct divisions within the human nucleus accumbens, which may be homologous to the 'shell'and'core'divisions of the nucleus as found in the rat.     

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.     

Inhibition of Brain Oestrogen Receptors by RU 58668

The purpose of this study was to determine if injections of the 11ß-substituted steroidal antioestrogen, RU 58668, would block two measures of oestrogen receptor action in ovariectomized adult female rats. Using an in vitro nuclear exchange assay, it was found that RU 58668 reduced cell nuclear []> ³H]-oestradiol binding in brain tissue 24  h after it was injected. However, pituitary cell nuclear [³H]-oestradiol binding was significantly reduced just 2  h after the antioestrogen was injected. Our results demonstrate that RU 58668 can reach the brain following a subcutaneous injection, but that it needs more time to reach the brain than it does to reach the pituitary. Since the levels of hypothalamic and pituitary progestin receptors are known to be regulated by oestradiol, cytosolic [³H]-R5020 binding was used as an in vitro assay of endogenous oestrogen receptor action. RU 58668 blocked induction by oestradiol of cytosolic [³H]-R5020 binding in both the brain and pituitary 48  h after it was injected. In the absence of oestradiol, RU 58668 did not stimulate cell nuclear [³H]-oestradiol binding or cytosolic [³H]-R5020 binding in either brain or pituitary. In conclusion, injections of RU 58668 blocked two measures of oestrogen receptor action in the brain and pituitary without showing oestrogenic activity itself.     

Distribution of Nicotinic Receptors in the Human Hippocampus and Thalamus

Neuronal nicotinic acetylcholine receptors consist of different subunits, α and β, with different subtype arrangement corresponding to distinct pharmacological and functional properties. The expression of α3, α7 and β2 mRNA in the human brain was studied by in situ hybridization and compared to [³H]nicotine, [³H]cytisine and [¹²⁵l]α-bungarotoxin binding in contiguous sections. The β2 probe showed a strong hybridization signal in the granular layer of the dentate gyrus and in the CA2/CA3 region of the hippocampus and in the insular cortex, and a signal of lower intensity in the subicular complex and entorhinal cortex. The α3 probe showed strong hybridization in the dorsomedial, lateral posterior, ventroposteromedial and reticular nuclei of the thalamus, and a weak signal in the hippocampal region and in the entorhinal, insular and cingular cortex. The amount of α7 mRNA was high at the level of the dentate granular layer and the CA2/CA3 region of the hippocampus, in the caudate nucleus and in the pulvinar and ventroposterolateral nuclei of the thalamus. [³H]Nicotine and [³H]cytisine binding appeared to be identical in anatomical distribution and relative intensity. It was high in the thalamic nuclei, the putamen and in the hippocampal formation in the subicular complex and the stratum lacunosum moleculare. The level of [¹²⁵l]α-bungarotoxin binding was particularly high in the hippocampus and in the pyramidal cells of the CA1 region, but was relatively low in the subicular complex. Our data indicate that in the human brain nicotinic receptor subtypes have discrete distributions, which are in part different from those of other species.     

Time Course of Dopamine-D2 and Serotonin-5-HT2 Receptor Occupancy Rates by Haloperidol and Clozapine In Vivo

Abstract: In vivo occupancy of dopamine-D₁, D₂ and serotonin-5-HT₂ receptors by haloperidol 10 mg/kg and clozapine 20 mg/kg were studied. Rats were injected intravenously with [³H]-YM-09151-2, [³H]-SCH23390, or [³H]-ketanserin 10 min after the administration of the tested drugs. Fifteen to 240 min after the ligand injection, the receptor occupancy rates of the drugs in the striatum and frontal cortex were calculated. Clozapine demonstrated the higher 5-HT₂ and lower D₂ occupancies in the respective regions. A dose-response analysis of D₂ and 5-HT₂, receptor occupancy by the drugs consolidated the higher 5-HT₂ binding affinity of clozapine in comparison with haloperidol. The present methodology may serve as an accurate tool to evaluate the peculiarity of various antipsychotics.     

Transferrin receptors in the normal human hippocampus and in Alzheimer's disease

Several lines of evidence suggest that aluminium may play a role in the pathogenesis of Alzheimer's disease (AD). The iron transport protein transferrin is the major transport protein for aluminium, and aluminium gains access to cells by means of a specific cell surface transferrin receptor. We have assessed the distribution of transferrin receptors in the normal and AD hippocampal formation using [³H]–transferrin ([³H]–Tf) binding and tritium film autoradiography, in order to assess the role of the transferrin receptor in AD. In normal brain, ³H]–Tf binding was highest in the pyramidal cell layers with CA2> dentate gyrus granule cell layer ≥Cal >CA3 ≥ CA4>subiculum>parahippocampal gyrus. In AD, significant reductions in [³H]–Tf binding were found in CA1, CA2 and CA4 pyramidal cell layers. The reduced [³H]–Tf binding in AD may, however, be due to poor pre–mortem agonal states which correlated with reduced [³H]–Tf binding. The discrepancy between the distribution of transferrin receptors in the hippocampus and those areas which are prone to the formation of senile plaques and neurofibrillary tangles suggests that if transferrin–mediated uptake of aluminium in AD/SDAT is significant in the pathogenesis of this disorder, it is not the only determinant of Alzheimer–type neuropathology.     

Plasticity of &mgr; and delta Opioid Receptors in the Superficial Dorsal Horn of the Adult Rat Spinal Cord Following Dorsal Rhizotomies: A Quantitative Autoradiographic Study

The aim was to study the regulation of μ and δ opioid binding sites in the superficial layers (laminae I–II) of the dorsal horn of the adult rat spinal cord 1, 2, 4 and 12 weeks after unilateral dorsal rhizotomies of various extents. Using quantitative autoradiography and highly selective tritiated opioid ligands, we have shown that the decrease in [³H]Tyr*- d -Ala-Gly-NMe-Phe-Gly-ol ([³H]DAMGO) (μ sites) and [³H]Tyr*- d -Thr-Gly-Phe-Leu-Thr ([³H]DTLET) (δ sites) binding in the side ipsilateral to the lesion as compared to the intact side is related to the number of dorsal roots cut. In the segment central to the lesion, 1 week after the lesion, ipsilateral/contralateral side binding ratios for [³H]DAMGO were 0.70, 0.49, 0.36 and 0.25 when 1, 3, 5 and 7 roots respectively were sectioned. For [³H]DTLET, the ratios were 0.71, 0.54, 0.42 and 0.39. The time-related analysis of binding ratios showed that, in partially deafferented spinal segments after long-term deafferentation (12 weeks postlesion) there were greater numbers of μ and δ binding sites than in cases of short-term deafferentation (1–2 weeks). By contrast, in spinal segments considered as completely deafferented, there was no difference in the remaining μ and δ binding sites at 12 weeks as compared to 1 week postlesion. Consequently, it is deduced that the partial recovery of μ and δ binding observed after long-term partial deafferentation could be associated with neuronal plasticity (probably collateral sprouting) of fine diameter primary afferent fibres arising from intact dorsal roots.     

Neurochemical aspects of the ontogenesis of cholinergic neurons in the rat brain

Ontogenic development of central cholinergic neurons in rat brain was examined by measuring the activity of choline acetyltransferase (CAT), concentration of acetylcholine (ACh) after focused microwave irradiation, the activity of the high affinity uptake process for choline and the apparent muscarinic receptor as quantified by specific binding of [³H]3-quinuclidinyl benzilate (QNB). For whole brain, the specific activity of CAT increases from 1 to 8% of adult between 15 days gestation and 7 days postpartum and then increases linearly to 83% by 4 weeks postpartum. The concentration of ACh is 22% of adult at 15 days gestation, rises to 29% by birth and attains adult levels by 4 weeks postpartum. The developmental rise in specific binding of [³H]QNB is intermediate between CAT and ACh with 10% of adult concentration of receptor at birth and a linear increase to 90% by 4 weeks postpartum. The development of the uptake of [³H]choline parallels that of CAT. In all regions of the neonatal rat brain, the relative level (% adult) of ACh is higher than [³H]QNB binding, which is higher than CAT. The neonatal medulla-pons has higher levels of [³H]QNB binding and activity of CAT (% adult) and develops more rapidly than the parietal cortex and corpus striatum; the hypothalamus and midbrain-thalamus exhibit intermediate rates of development.     

β-Carbolines: Endogenous Ligands for the Benzodiazepine Receptor

Abstract: The interaction of benzodiazepines (BDZ) and β -carbolines with metal cations was investigated. Δmong the numerous transition metal cations, only four, Co²⁺, Ni², Cu²⁺ and Zn²⁺, specifically affected the receptor binding of [³H]diazepam and [³H] β -carboline-3-carboxylic acid ethyl ester ( β -CCE). The effects of these cations on [³H]diazepam binding were exactly opposite to those on [³H] β -CCE binding. An intraperitoneal injection of β -carboline-3-carboxylic acid methyl ester ( β -CCM) produced spike discharges and β-CCM induced spike discharges were prevented by an intramuscular injection of BDZ. These findings suggest that β -carbolines could be related to the proposed endogenous ligand and that BDZ might be antagonistic rather than agonistic of the naturally occurring ligand.     

Downregulation of muscarinic receptors in the rat caudate-putamen after lesioning of the ipsilateral nigrostriatal dopamine pathway with 6-hydroxydopamine (6-OHDA): normalization by fetal mesencephalic transplants

The autoradiographic distribution and density of muscarinic receptors was studied in the neostriatum of rats with long-term unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal dopaminergic pathway and in lesioned rats who had additionally received embryonic substantia nigra grafts in the dopamine denervated striatum. Muscarinic receptors were labeled with [³H]quinuclidinyl benzilate (QNB), M₁ receptors were directly labeled with [³H]pirenzepine (PZ) and non-M₁ receptors were labeled by the competition of 100 nM PZ with [³H]QNB. The density and distribution of muscarinic receptors were directly compared to the sodium-dependent, high-affinity, choline uptake sites as labeled with [³H]hemicholinium-3 (HC-3). In the 6-OHDA-lesioned animals, there was a 25% reduction in muscarinic receptors labeled with [³H]QNB. Subtype analysis showed that there was a reduction of both M₁ (−26%) and non-M₁ (−33%) receptors. A normal density of both muscarinic receptor populations was found in animals with successful transplants. Saturation analysis demonstrated that the changes, in muscarinic receptor density, were due to a change in receptor number (B_max) and not affinity (K_d). There was no significant change in [³H]HC-3 binding in the 6-OHDA-lesioned or transplanted animals, indicating that alterations in muscarinic receptors were not due to transynaptic degeneration of striatal cholinergic interneurons. The findings of downregulation of muscarinic receptors following long-term dopamine denervation and the subsequent normalization of muscarinic receptor density after fetal mesencephalic transplantation suggests that transplanted substantia nigra cells are able to restore inhibitory control on striatal cholinergic interneurons.     

[3H]Nitrendipine Binding to Rabbit Colonic Smooth Muscle

We used [³H] nitrendipine binding to isolated smooth muscle cells and isometric tension studies of muscle strips to characterize the calcium channels from rabbit proximal and distal colon. At 25°C [³H] nitrendipine binding was rapid, saturable, reversible, specific, and linearly proportional to cell number. The affinity of the ligand for its receptor was similar in proximal and distal colon (K_D 129 ± 21 pM and 124 ± 17 pM, respectively). In the proximal colon there were 68,000 receptors per cell, compared to 58,000 receptors per cell in the distal colon (p > .1). The Hill coefficient for nitrendipine was close to unity, suggesting binding to a single receptor. Although nitrendipine and nifedipine competitively inhibited [³H]nitrendipine binding, verapamil did not alter [³H] nitrendipine binding, suggesting the presence of at least two discrete, noninteracting sites for the binding of drugs that block calcium channels. In studies with muscle strips nitrendipine competitively inhibited isometric tension stimulated by both bethanechol and high extracellular potassium concentration. There were no significant differences in response from proximal and distal colon. These results suggest that calcium antagonist binding characteristics to calcium channels are similar in proximal and distal colon, and do not explain previously observed differences in the function of muscle in these tissues.     

Kindling-Induced Changes of GTP Binding in the Cerebral Cortical Membrane

Abstract: Specific [³H]GTP binding to the cerebral cortical membrane was examined in amygdaloid kindled rats. Membrane fractions of the cerebral cortex obtained from kindled rats and sham operated control were incubated with [³H]GTP and the data were analyzed by Scatchard plots. There were no differences in the B_max and K_d values in basal (without any agonists) binding between kindled and control membranes. In the presence of isoprenaline, β-agonist, B_max values increased in the control, but did not increase in the kindled group. The kindling-induced abolishment of isoprenaline effect on B_max of GTP binding persisted at least two weeks. Specific GTP binding activity of control membrane increased in the presence of carbachol, muscarinic agonist. A carbachol-induced increase in specific GTP binding activity also disappeared in the kindled group. These results suggest that a functional imbalance among various types of GTP binding proteins (Gs, Gi or Go) might be related to the acquisition of the epileptogenesis in the kindling model of epilepsy.     

Selective alterations in glutamate receptor subtypes after unilateral orbital enucleation

Glutamate is the major excitatory neurotransmitter in the rat visual system. Using quantitative autoradiography the effect of unilateral orbital enucleation on [³H]kainate, [³H]-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid ([³H]AMPA) and [³H]glutamate binding to kainate, quisqualate and NMDA receptors respectively has been examined within anatomical components of the visual pathway at 4 time points up to 20 days post-lesion. The time course for the degeneration of retinal projection fibres was assessed in a separate group of animals by quantifying [³H]cyclohexyladenosine ([³H]CHA) binding to presynaptic adenosine A₁ receptors. Over the first 5 days after orbital enucleation, there were no significant alterations in glutamate or adenosine A₁ receptor binding in visual structures of the visually deprived hemisphere. However, at 10 days post-lesion [³H]AMPA binding was significantly reduced (30%) in the visually deprived superior colliculus but unaltered in other visual structures. At this time point there was also a significant reduction (50%) in [³H]CHA binding in the visually deprived superior colliculus but not in other retino-recipient nuclei. There were similar changes in [³H]AMPA and [³H]CHA binding at 20 days post-enucleation. [³H]Kainate binding was significantly increased in the visually deprived superior colliculus only at 20 days post-enucleation. Saturation analysis of [³H]kainate and [³H]AMPA binding at this time point indicated a selective increase in the b_max value for the high affinity [³H]kainate binding site and a concomitant decrease in the b_max value for the high affinity [³H]AMPA binding site in the visually deprived superior colliculus. There were, however, no significant alterations in [³H]AMPA or [³H]kainate binding in other primary projection areas or in secondary visual areas (e.g. visual cortex) at any time point. NMDA sensitive [³H]glutamate binding was unaltered in the visually deprived hemisphere up to 20 days post-enucleation. These results suggest an upregulation of kainate receptors in the visually deprived superior colliculus after orbital enucleation and a loss of presynaptic quisqualate receptors on degenerating retinal fibres. The plastic alterations in kainate receptors in the superior colliculus are supportive of electrophysiological data suggesting a physiological role for these sites in mediating excitatory postsynaptic potentials in tectal neurons.