B. Collier

Localization of interleukin-2 immunoreactivity and interleukin-2 receptors in the rat brain: interaction with the cholinergic system

The present work characterizes the presence of interleukin-2 (IL-2)-like immunoreactive material and IL-2 binding sites in the adult rat brain. The results show that there are detectable levels of IL-2-like material in extracts of rat hippocampus, striatum, and frontal cortex. However, specific [125I]IL-2 binding sites were observed only in the hippocampus, using both homogenate-binding and autoradiographic techniques. In this region of the rat brain, specific [125I]IL-2 binding was inhibited by 100 nM non-radioactive recombinant human IL-2. In kainate-lesioned hippocampi, the density of [125I]IL-2 sites was apparently increased, suggesting their localization to extrinsic innervation and/or glial cells. In slices of hippocampus, which contain both IL-2-like immunoreactive material and specific IL-2 sites, exogenous IL-2 significantly decreased the potassium (25 mM)-evoked, but not the basal, release of acetylcholine. This IL-2-induced effect was concentration-dependent, and was apparent at a relatively low concentration (1 nM). This IL-2 effect was also region-specific, such that acetylcholine release from other tissue slices (striatal, frontal cortical) was not affected. In slices from kainate-lesioned hippocampi, the IL-2-induced reduction of acetylcholine release was only modestly enhanced, suggesting that the extra IL-2 sites that appear post-lesion may not be localized to cholinergic terminals.

Amyloid β-Peptide Inhibits High-Affinity Choline Uptake and Acetylcholine Release in Rat Hippocampal Slices

Abstract: The characteristic pathological features of the postmortem brain of Alzheimers disease (AD) patients include, among other features, the presence of neuritic plaques composed of amyloid β‐peptide (Aβ) and the loss of basal forebrain cholinergic neurons, which innervate the hippocampus and the cortex. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that Aβ accumulation in vivo may initiate and/or contribute to the process of neurodegeneration and thereby the development of AD. However, the mechanisms by which Aβ peptide influences/causes degeneration of the basal forebrain cholinergic neurons and/or the cognitive impairment characteristic of AD remain obscure. Using in vitro slice preparations, we have recently reported that Aβ‐related peptides, under acute conditions, potently inhibit K+‐evoked endogenous acetylcholine (ACh) release from hippocampus and cortex but not from striatum. In the present study, we have further characterized Aβ‐mediated inhibition of ACh release and also measured the effects of these peptides on choline acetyltransferase (ChAT) activity and high‐affinity choline uptake (HACU) in hippocampal, cortical, and striatal regions of the rat brain. Aβ1–40 (10−8M) potently inhibited veratridine‐evoked endogenous ACh release from rat hippocampal slices and also decreased the K+‐evoked release potentiated by the nitric oxide‐generating agent, sodium nitroprusside (SNP). It is interesting that the endogenous cyclic GMP level induced by SNP was found to be unaltered in the presence of Aβ1–40. The activity of the enzyme ChAT was not altered by Aβ peptides in hippocampus, cortex, or striatum. HACU was reduced significantly by various Aβ peptides (10−14 to 10−6M) in hippocampal and cortical synaptosomes. However, the uptake of choline by striatal synaptosomes was altered only at high concentration of Aβ (10−6M). Taken together, these results indicate that Aβ peptides, under acute conditions, can decrease endogenous ACh release and the uptake of choline but exhibit no effect on ChAT activity. In addition, the evidence that Aβ peptides target primarily the hippocampus and cortex provides a potential mechanistic framework suggesting that the preferential vulnerability of basal forebrain cholinergic neurons and their projections in AD could relate, at least in part, to their sensitivity to Aβ peptides.

Insulin-like growth factor-1 (somatomedin-C) receptors in the rat brain: distribution and interaction with the hippocampal cholinergic system

The present work characterizes the autoradiographic distribution of insulin-like growth factor-1 (IGF-1)/somatomedin-C binding sites in neonatal and adult rat brain, and attempts to correlate the distribution of IGF-1 sites, in certain regions of the rat brain, with functional IGF-1 receptors. In neonatal brain, [125I]IGF-1 binding sites are especially concentrated in superficial cortical layers, nucleus accumbens and hippocampus. In the adult rat brain, the distribution of IGF-1 sites is broader, with a high density of sites observed in superficial and deep cortical layers, olfactory bulb, endopiriform nucleus, basomedial nucleus of the amygdala, thalamic nuclei and hippocampus. Specific binding of [125I]IGF-1 to its sites in these brain regions was almost completely inhibited by 100 nM nonradioactive IGF-1. In contrast, similar concentrations of either IGF-2 or insulin did not significantly alter [125I]IGF-1 binding to its sites. Therefore, under our incubation conditions, [125I]IGF-1 appears to label specifically the type-I IGF receptor. In the hippocampus, which is highly enriched with specific [125I]IGF-1 binding sites in both neonatal and adult rat brain, IGF-1 significantly altered the potassium-evoked (25 mM) release of acetylcholine (ACh) from slices of adult, but not immature (6- and 18-day-old), rat brain. This IGF-1-induced decrease in ACh release from adult rat brain slices was concentration-dependent and appeared to be specific to hippocampus; ACh release from frontal cortical slices was not affected by this GF. The spontaneous release of ACh in the presence of IGF-1 in either tissue was not significantly different from control.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of endogenous acetylcholine release from mammalian brain slices by opiate receptors: hippocampus, striatum and cerebral cortex of guinea-pig and rat.

The effects of opiate agonists on acetylcholine release from hippocampal, striatal and cerebral cortical slices were tested; tissue from rat was compared to that from guinea-pig. The results show that opiate receptors in each of these areas can alter the evoked release of acetylcholine from nerve terminals; however, there are species and tissue differences with respect to the apparent subtype of opiate receptor effective. In the hippocampus and striatum of the two species studied, opiates caused a dose-dependent decrease in evoked acetylcholine release from tissue slices but in the guinea-pig kappa-selective agonists were effective, and mu or delta agonists were not, whereas in the rat, mu-, but not delta- or kappa-selective drugs were effective. Opiates also altered acetylcholine release from the frontal, parietal and occipital cortex of both of these species. In all three regions of the guinea-pig cortex, kappa and delta agonists were active and in the parietal cortex mu agonists were also active; rat cortical slices showed similar results except that delta agonists were not effective. The inhibitory effects of the opiate agonists were effectively antagonized by the non-selective opiate antagonist naloxone and by the calcium channel agonists, BAY K 8644 or YC-170. In addition, the effects of the opiate drugs tested in this study on acetylcholine release were confined to evoked release, that is, spontaneous acetylcholine release was not affected. The results suggest that in guinea-pig and rat brain, opiate receptors regulate acetylcholine release, and that, although the subtypes of opiate receptors involved in this effect are different in the two species and in different tissues from the same species, the effect results from a common mechanism that involves alterations of calcium influx into the nerve terminals during depolarization.

Characterization of N-[3H]methylcarbamylcholine binding sites and effect of N-methylcarbamylcholine on acetylcholine release in rat brain

The present experiments show that N‐[3H]‐methylcarbamylcholine ([3H]MCC) binds specifically and with high affinity to rat hippocampus, frontal cortex, and striatum. The highest maximal density of binding sites was apparent in frontal cortex and the lowest in hippocampus. [3H]MCC binding was potently inhibited by nicotinic, but not muscarinic, agonists and by the nicotinic antagonist dihydro‐β‐erythroidine in all three brain regions studied. The effect of unlabeled MCC on acetylcholine (ACh) release from slices of rat brain was tested. The drug significantly enhanced spontaneous ACh release from slices of hippocampus and frontal cortex, but not from striatal slices. This effect of MCC to increase ACh release from rat hippocampus and frontal cortex was antagonized by the nicotinic antagonists dihydro‐β‐erythroidine and d‐tubocurarine, but not by α‐bungarotoxin or by the muscarinic antagonist atropine. The MCC‐induced increase in spontaneous ACh release from hippocampal and frontal cortical slices was not affected by tetrodotoxin. The results suggest that MCC might alter cholinergic transmission in rat brain by a direct activation of presynaptic nicotinic receptors on the cholinergic terminals. That this alteration of ACh release is apparent in hippocampus and frontal cortex, but not in striatum, suggests that there may be a regional specificity in the regulation of ACh by nicotinic receptors in rat brain.

Binding sites for [3H]AF-DX 116 and effect of AF-DX 116 on endogenous acetylcholine release from rat brain slices

The present study shows that the putative M2 ligand, [3H]AF-DX 116, binds to two classes of muscarinic sites in homogenates of rat hippocampus, striatum and cerebral cortex: one with a high affinity (Kd less than 5 nM)/low capacity (Bmax = 30-63 fmol/mg protein), and a second of lower affinity (Kd greater than 65 nM) and higher capacity (Bmax greater than 190 fmol/mg protein). In experiments which tested the effects of the muscarinic antagonists on acetylcholine (ACh) release from brain slices, the non-selective antagonist (-)-quinuclidinyl benzylate and atropine significantly enhanced the potassium (25 mM)-evoked release of ACh. This effect was mimicked by the M2 ligand AF-DX 116, but neither the M1-selective antagonist pirenzepine, nor the putative M3-muscarinic antagonist, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), altered ACh release. Also, the muscarinic agonist, oxotremorine, significantly depressed evoked ACh release from brain slices, an effect that was completely antagonized by atropine or by AF-DX 116, but not by pirenzepine or 4-DAMP. Thus, it appears that presynaptic muscarinic autoreceptors in the rat hippocampus, striatum and cerebral cortex belong to the M2 subtype of muscarinic receptors.

Effect of chronic nicotine treatment on nicotinic autoreceptor function and N-[3H]methylcarbamylcholine binding sites in the rat brain

Abstract: It has been reported that N‐methylcarbamylcholine (MCC), a nicotinic agonist, binds to central nicotinic receptors and causes an increase of acetylcholine (ACh) release from certain central cholinergic nerve terminals. The present experiments determine whether these two phenomena change in response to the chronic administration of nicotine, a procedure known to result in an increase in nicotinic binding sites. Chronic nicotine caused a brain region‐specific up‐regulation of [3H]MCC sites; binding increased in the frontal cortex, parietal cortex, striatum, and hippocampus, but not in the occipital cortex or cerebellum. The effect of nicotine was selective to nicotinic binding sites, because muscarinic sites, both M1 ([3H]pirenzepine) and M2 ([3H]ACh), were unaffected by chronic nicotine treatment. MCC increased the release of ACh from the frontal cortex and hippocampus by a calcium‐dependent mechanism; MCC did not alter ACh release from striatum or occipital cortex of control animals. The MCC‐induced increase in ACh release was not apparent in those animals which had been treated with nicotine. There was a partial recovery of nicotinic autoreceptor function when animals were allowed to recover (4 days) following chronic nicotine treatment, but the density of binding sites remained increased compared to control. Chronic nicotine did not change the potassium‐evoked release of ACh from the frontal cortex or hippocampus, but decreased this measure from striatum. It also decreased the ACh content of the striatum, but not that of the cortex or the hippocampus; the activity of choline acetyltransferase was not altered in any of the regions tested. These results suggest that chronic nicotine increases the nicotinic ([3H]MCC) binding site density in the frontal cortex, hippocampus, and striatum and results in the loss of nicotinic receptor‐mediated positive feedback in the cortex and hippocampus. The loss of presynaptic nicotinic receptor function is reversible, and the time course of this recovery differs from that of the change in [3H]MCC binding sites.

Cell Growth Inhibition and Functioning of Human Somatostatin Receptor Type 2 Are Modulated by Receptor Heterodimerization

Somatostatin (SST) analogs have been successfully used in the medical treatment of acromegaly, caused by GH hypersecreting pituitary adenomas. Patients on SST analogs rarely develop tachyphylaxis despite years of continuous administration. It has been recently proposed that a functional association between SST receptor (SSTR) subtypes 2 and 5 exists to account for this behavior; however, a physical interaction has yet to be identified. Using both coimmunoprecipitation and photobleaching fluorescence resonance energy transfer microscopy techniques, we determined that SSTR2 and SSTR5 heterodimerize. Surprisingly, selective activation of SSTR2 and not SSTR5, or their costimulation, modulates the association. The SSTR2-selective agonist L-779,976 is more efficacious at inhibiting adenylate cyclase, activating ERK1/2, and inducing the cyclin-dependent kinase inhibitor p27(Kip1) in cells expressing both SSTR2 and SSTR5 compared with SSTR2 alone. Furthermore, cell growth inhibition by L-779,976 treatment was markedly extended in coexpressing cells. Trafficking of SSTR2 is also affected upon heterodimerization, an attribute corresponding to modifications in beta-arrestin association kinetics. Activation of SSTR2 results in the recruitment and stable association of beta-arrestin, followed by receptor internalization and intracellular receptor pooling. In contrast, heterodimerization increases the recycling rate of internalized SSTR2 by destabilizing its interaction with beta-arrestin. Given that SST analogs show preferential binding to SSTR2, these data provide a mechanism for their effectiveness in controlling pituitary tumors and the absence of tolerance seen in patients undergoing long-term administration.

THE FORMATION OF CHOLINE AND OF ACETYLCHOLTNE BY BRAIN IN VITRO

Abstract— Free choline and acetylcholine (ACh) in mouse or rat brain were assayed biologically. The subcellular distribution of ACh in brain slices that had been incubated in the presence of eserine was compared to that in control brain; during incubation, the ACh outside nerve endings increased four‐fold, the ACh released from synaptosomes by osmotic shock doubled but the ACh bound firmly within nerve endings did not increase. The two nerve ending stores of ACh were labelled to similar specific radioactivities when slices were incubated with [3H]choline, but the specific radioactivity of the ACh formed was much lower than that of the added choline. Tissue incubated in the presence of eserine released choline and ACh into the medium and the tissue levels of both substances increased. Brain tissue exposed to Na+‐free medium lost 84 per cent of its ACh and 66 per cent of its free choline; the amounts of both substances returned towards control values during subsequent incubation in a normal‐Na+ medium (choline‐free). Both the ACh outside nerve endings and the ACh associated with synaptosomes were depleted when tissue was incubated in Na+‐free medium.

Morphine-Induced Changes in δ Opioid Receptor Trafficking Are Linked to Somatosensory Processing in the Rat Spinal Cord

An in vivo fluorescent deltorphin (Fluo-DLT) internalization assay was used to assess the distribution and regulation of pharmacologically available δ opioid receptors (δORs) in the rat lumbar (L4-5) spinal cord. Under basal conditions, intrathecal injection of Fluo-DLT resulted in the labeling of numerous δOR-internalizing neurons throughout dorsal and ventral horns. The distribution and number of Fluo-DLT-labeled perikaryal profiles were consistent with that of δOR-expressing neurons, as revealed by in situ hybridization and immunohistochemistry, suggesting that a large proportion of these cells was responsive to intrathecally administered δOR agonists. Pretreatment of rats with morphine for 48 hr resulted in a selective increase in Fluo-DLT-labeled perikaryal profiles within the dorsal horn. These changes were not accompanied by corresponding augmentations in either δOR mRNA or 125I-deltorphin-II binding levels, suggesting that they were attributable to higher densities of cell surface δOR available for internalization rather than to enhanced production of the receptor. Unilateral dorsal rhizotomy also resulted in increased Fluo-DLT internalization in the ipsilateral dorsal horn when compared with the side contralateral to the deafferentation or to non-deafferented controls, suggesting that δOR trafficking in dorsal horn neurons may be regulated by afferent inputs. Furthermore, morphine treatment no longer increased Fluo-DLT internalization on either side of the spinal cord after unilateral dorsal rhizotomy, indicating that μOR-induced changes in the cell surface availability of δOR depend on the integrity of primary afferent inputs. Together, these results suggest that regulation of δOR responsiveness through μOR activation in this region is linked to somatosensory information processing.