Changes in postnatal norepinephrine alter alpha-2 adrenergic receptor development.

Alpha-2 adrenergic receptors (A2AR) regulate multiple brain functions and are enriched in developing brain. Studies demonstrate norepinephrine (NE) plays a role in regulating brain maturation, suggesting it is important in A2AR development. To investigate this we employed models of NE absence and excess during brain development. For decreases in NE we used N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP4), a specific noradrenergic neurotoxin. Increased noradrenergic terminal density was produced by methylazoxymethanol acetate (MAM) treatment. A2AR density was assayed with [(3)H]RX821002 autoradiography. DSP4 lesions on postnatal day (PND) 3 produce A2AR decreases in many regions by PND 5. A2AR recover to control levels by PND 15 and 25 and there is no further change in total receptor density. We also assayed A2AR in brains lesioned with DSP4 on PND 13, 23, 33 and 43 and harvested 22 days post-lesion. A2AR levels remain similar to control at each of these time points. We examined A2AR functionality and high affinity state with epinephrine-stimulated [(35)S]GTPγS and [(125)I]p-iodoclonidine autoradiography, respectively. On PND 25, control animals and animals lesioned with DSP4 on PND 3 have similar levels of [(35)S]GTPγS incorporation and no change in high affinity state. This is in contrast to increases in A2AR high affinity state produced by DSP4 lesions of mature brain. We next investigated A2AR response to increases in norepinephrine levels produced by MAM. In contrast to DSP4 lesions, increasing NE results in a large increase in A2AR. Animals treated with MAM on gestational day 14 had cortical [(3)H]RX821002 binding 100-200% greater than controls on PND 25, 35, 45, 55 and 65. These data indicate that NE regulation of A2AR differs in developing and mature brain and support the idea that NE regulates A2AR development and this has long term effects on A2AR function.

Differential effects of neonatal norepinephrine lesions on immediate early gene expression in developing and adult rat brain.

Activity regulated cytoskeletal protein (Arc), c-fos and zif268 are immediate early genes (IEGs) important for adult brain plasticity. We examined developmental expression of these IEGs and the effect of neonatal noradrenergic lesion on their expression in developing and mature brain. N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4), a specific noradrenergic neurotoxin, was administered to rats on postnatal day (PND) 3 and in situ hybridization was used to assay Arc, c-fos and zif268 mRNA on PND 13, 25 and 60. In contrast to decreases in Arc, c-fos and zif268 expression produced by noradrenergic lesions of mature brain, lesions on PND 3 yield a strikingly different effect. Neonatal lesions produce increases in c-fos and zif268 expression in specific frontal cortical layers on PND 13, while Arc shows no change. These lesions lead to increases in zif268 expression in frontal cortical layers on PND 25, with no changes in c-fos or Arc expression, and on PND 60 they produce a significant increase in c-fos expression in hippocampus with no significant changes in Arc or zif268 expression. 2-[2-(2-Methoxy-1,4-benzodioxanyl)]imidazoline hydrochloride (RX821002), an alpha-2 adrenergic receptor (A2AR) antagonist, administered to control PND 60 animals produces elevations of Arc, zif268 and c-fos mRNAs. This response was eliminated in animals lesioned with DSP-4 on PND 3. These data indicate that norepinephrine regulation of IEG expression differs in developing and mature brain and that loss of developmental norepinephrine leads to abnormally high postnatal IEG expression. Previous studies have shown an important role for norepinephrine in brain development. Our data support the idea that norepinephrine plays an important role during CNS development and that changes in noradrenergic signaling during development may have long lasting effects, potentially on learning and memory.

Development of the norepinephrine transporter in the rat CNS.

The norepinephrine transporter (NET) plays a major role in regulating the actions of norepinephrine by removing norepinephrine from the synapse. Many studies suggest norepinephrine plays an important role in regulating development of the CNS, pointing to NET as an important factor in this process. We examined the ontogeny of NET expression in rat brain at 5, 10, 15, 20 and 25 days postnatally (PND) and in adults, using quantitative autoradiography with [3H]nisoxetine as ligand. At PND 5 and 10 most forebrain areas had low NET expression (1-2 fmol/mg tissue). By PND 15 most forebrain areas increased NET expression approximately five-fold compared with PND 10, levels generally similar to those found in the adult brain. In contrast, NET development in the brainstem exhibited elevated densities at PND 5, 10 and 20 that decreased in the adult. The locus coeruleus, in particular, had very high NET expression in the early postnatal period that decreased dramatically in the adult brain. These data illustrate a dynamic ontogenic profile for NET, characterized by developmental increases in forebrain structures and contrasting decreases in the brainstem. The early postnatal expression of NET in brainstem and the subsequent decrease or loss of NET expression with maturation suggest an important role for this transporter and for norepinephrine in the development of many brain regions. These studies also have important implications for use of drugs targeting the noradrenergic system in children and adolescents, such as antidepressants and drugs of abuse.

Alpha-2 adrenergic receptor development in rat CNS: an autoradiographic study.

During development norepinephrine plays a role in determining the morphologic organization of the CNS and the density and future responsiveness of adrenergic receptors. alpha-2 Adrenergic receptors, one of three adrenergic receptor types, regulate important adult CNS functions and may have a distinct role during development. We examined alpha-2 receptor distribution and density in the rat brain at postnatal days 1, 5, 10, 15, 21, 28 and in adults using the antagonist [(3)H]RX821002 for autoradiography. Binding kinetics and pharmacology for alpha-2 adrenergic receptors were the same in adults and neonates. There was an overall increase in alpha-2 receptor levels during postnatal development with great variability in pattern and timing of receptor density changes among brain regions. Three major patterns were apparent. First, in many regions receptor density increased during postnatal development, generally reaching adult levels around postnatal day 15. Within this group there was variability in timing between regions and there were several regions with receptor densities higher than adult levels during the postnatal period. Second, there were regions with very high levels of receptors at birth and little or no change in density during the postnatal period. Third, some regions demonstrated decreasing or transient expression of alpha-2 adrenergic receptors in the course of postnatal development, including white matter regions, cerebellum and many brainstem nuclei, suggesting specific roles for alpha-2 receptors during development. This study investigates the development of alpha-2 adrenergic receptors in the rat CNS. It demonstrates there is region-specific regulation of alpha-2 receptor development and identifies brain regions where these receptors may play a specific and critical role in the regulation normal development.

Agonist-stimulated [35S]GTPgammaS autoradiography: optimization for high sensitivity.

The receptor-stimulated accumulation of [35S]GTPgammaS provides a measure of functional coupling of G proteins with receptors. Sensitivity for autoradiographic visualization of [35S]GTPgammaS binding was improved two- to threefold in rat brain sections by optimizing assay conditions. Non-specific (NSB), basal and agonist-stimulated [35S]GTPgammaS binding were measured, using methadone, 5-carboxamidotryptamine and epinephrine for mu-opiate receptors, 5-HT(1A) receptors and alpha(2)-adrenoceptors. Basal and NSB were low in glycylglycine buffer compared to Tris or HEPES buffers, and agonist-stimulated [35S]GTPgammaS binding was more easily observed. Further optimization using glycylglycine buffer found increased signal-to-noise ratio with inclusion of dithiothrietol, increased [35S]GTPgammaS incubation time (2-4 h) and guanosine 5'-diphosphate (GDP) preincubation (20-30 min), and use of [35S]GTPgammaS at 0.1 nM. Improved sensitivity was due to decreased NSB and basal [35S]GTPgammaS binding and agonist-stimulated binding were similarly affected for each receptor system. The assay conditions described should extend the use of agonist-stimulated [35S]GTPgammaS autoradiography to receptors, which produce low levels of [35S]GTPgammaS binding and to the measurement of changes in receptor-G protein coupling.

A robust GTP-induced shift in alpha(2)-adrenoceptor agonist affinity in tissue sections from rat brain.

A method is presented for monitoring the coupling of the alpha(2)-adrenoceptor, as well as other receptors, to their G proteins using the GTP-induced shift in agonist affinity states. In tissue sections GTP, but not ATP, induces a robust decrease in agonist affinity of greater than 100-fold, which is much larger than previously found in membrane preparations. A sensitive and easy procedure to monitor the extent of coupling is to compare the amount of [(3)H]RX821002 binding remaining in the presence of 100 nM brimonidine in the absence and presence of 100 microM GTP. This method should be especially applicable for determining the extent of coupling of receptors to their G proteins in multiple brain regions using autoradiographic procedures.

Cholinergic receptors: dual roles in transduction and plasticity.

The regional distributions and possible functions of nicotinic acetylcholine receptors (nAChRs) in the developing and adult auditory rat brain are reviewed. The predominant nAChR in the auditory brainstem is the alpha7 homomeric receptor. alpha7 mRNA and protein are expressed in selected regions of the cochlear nucleus (CN), inferior colliculus (IC), medial superior olive, lateral superior olive, ventral nucleus of the lateral lemniscus and superior paraolivary nucleus. Peak expression of mRNA and protein occurs by the second postnatal week in most auditory brainstem areas. In contrast, the alpha3 and beta4 nicotinic subunits are expressed in the embryo and early in postnatal development in the CN and IC, but not other brainstem nuclei. Of particular interest is the octopus cell region of the posteroventral cochlear nucleus (PVCN). alpha3 and beta4 are down-regulated in the octopus cell region about postnatal day 10, which is the age that alpha7 is at peak expression. NAChRs play important roles in transduction and in regulating intracellular calcium. The ability of the alpha7 receptor to synchronize synaptic activity and stabilize synapses makes it a prime candidate as a mechanism underlying homeostatic plasticity in the auditory system.

Alpha(2)-adrenoceptor-stimulated GTP gamma S binding in rat brain: an autoradiographic study.

Agonist-stimulated [35S]GTP gamma S binding by alpha(2)-adrenoceptors was examined in rat brain by autoradiography. Epinephrine, norepinephrine, dexmedetomidine and brimonidine stimulated [35S]GTP gamma S binding in a dose-dependent manner. Agonist-stimulated binding was blocked by the specific alpha(2)-adrenoceptor antagonist (1, 4-benzodioxan-2-methoxy-2-yl)-2-imidazoline hydrochloride (RX821002). Each alpha(2)-adrenoceptor agonist stimulated [35S]GTP gamma S binding in the same brain regions, corresponding to alpha(2)-adrenoceptor distribution determined by [125I]para-iodoclonidine autoradiography. The order of antagonist potency (RX821002>idazoxan>rauwolscine>phentolamine>prazosin), and weak inhibition by propranolol and selective serotonin antagonists, indicate that epinephrine-stimulated [35S]GTP gamma S binding is mediated primarily by alpha(2)-adrenoceptors. Several antagonists increased [35S]GTP gamma S binding at very high concentrations, and this effect had anatomic and pharmacologic characteristics of binding mediated by 5-HT(1A) receptors. These studies demonstrate functional linkage of alpha(2)-adrenoceptors to G proteins in tissue sections, thus providing data on neuroanatomic localization and a means to examine drug specificity at alpha(2)-adrenoceptors in different brain regions.

Inverse agonism at alpha(2)-adrenoceptors in native tissue.

Several alpha(2)-adrenoceptor antagonists have inverse agonist properties in cell culture systems, usually expressing high levels or a constitutively active form of alpha(2)-adrenoceptors. In characterizing the binding of alpha(2)-adrenoceptor agonists to rat brain tissue sections, we found that conditions known to alter agonist affinity for these receptors, particularly the addition of 100 microM GTP, altered the binding of the alpha(2)-adrenoceptor antagonist, [3H](1,4-benzodioxan-2-methoxy-2-yl)-2-imidazoline hydrochloride (RX821002). In further studies, we found that under our conditions [3H]RX821002 demonstrates inverse agonist properties at alpha(2)-adrenoceptors. This is the first demonstration of inverse agonism at alpha(2)-adrenoceptors in native tissue. We found that the alpha(2)-adrenoceptor antagonist, (2S,12bS)1', 3'-dimethylspiro(1,3,4,5',6,6',7,12b-octahydro-2H-benzo(b)fu ro(2, 3-a)quinazoline)-2,4'-pyrimidin-2'-one (MK-912), did not have clearly discernible inverse agonist properties and acted as a neutral antagonist in these studies. On the other hand, the antagonist rauwolscine actually displayed partial agonist properties in our studies. These findings indicate that the inverse agonist properties of alpha(2)-adrenoceptor antagonists can be demonstrated in native tissue, as well as in tissue culture, and they strengthen the idea that inverse agonist properties may be of physiological and pharmacological importance.

Alpha-2 adrenergic receptor functional coupling to G proteins in rat brain during postnatal development.

During postnatal development, alpha-2 adrenergic receptors (A2AR) change in both density and distribution. In forebrain, receptor density increases about 4-fold over neonatal levels, reaching adult levels before postnatal day (P) 28, whereas in hindbrain, including cerebellum, there is a decrease in overall receptor density. We examined the coupling of A2AR to G proteins using agonist-stimulated [35S]GTPgammaS binding as a functional assay. In forebrain the A2AR agonist-stimulated [35S]GTPgammaS binding increases rapidly after P7, reaching its highest levels at P21 and then declining slightly to adult levels. This binding increases more slowly than receptor number, suggesting that the appearance of G proteins, rather than the A2AR, determines the developmental appearance of functional A2AR-G protein interactions in forebrain. Basal [35S]GTPgammaS binding and [35S]GTPgammaS binding stimulated by other neurotransmitter receptor systems (GABA-B, mu opiate, and muscarinic) increase with a time course similar to A2AR-stimulated [35S]GTPgammaS binding. In contrast, in hindbrain, A2AR-stimulated [35S]GTPgammaS binding decreases during postnatal development in parallel with the decrease in A2AR levels, whereas [35S]GTPgammaS binding stimulated by other neurotransmitter receptor systems increases in parallel with basal [35S]GTPgammaS binding. Functional receptor-G protein coupling in hindbrain appears to be dependent on the developmental appearance of G proteins for most neurotransmitter systems. However, for A2AR the decrease in receptor density is the overriding factor. These studies 1) demonstrate the functional measurement of A2AR-G protein coupling in native tissue for the first time, 2) demonstrate that A2AR are coupled to G proteins throughout postnatal development, and 3) describe developmental increases and decreases in functional A2AR in brain.

Nicotinic acetylcholine receptors in rat cochlear nucleus: [125I]-alpha-bungarotoxin receptor autoradiography and in situ hybridization of alpha 7 nAChR subunit mRNA.

The cochlear nucleus (CN) is the first site in the central nervous system (CNS) for processing auditory information. Acetylcholine in the CN is primarily extrinsic and is an important neurotransmitter in efferent pathways thought to provide CNS modulation of afferent signal processing. Although muscarinic acetylcholine receptors have been studied in the CN, the role of nicotinic receptors has not. We examined the distribution of one nicotinic acetylcholine receptor subtype, the alpha-bungarotoxin receptor (alpha Bgt), in the CN. Quantitative autoradiography was used to localize receptors and in situ hybridization was used to localize alpha 7 mRNA in CN neurons that express the alpha Bgt receptor. Binding sites for alpha Bgt are abundant in the anterior ventral, posterior ventral, and dorsal divisions of the CN, and receptor density is low in the granule cell layer and interstitial nucleus. Heterogeneity in CN subregions is described. Four distinct patterns of alpha Bgt binding were observed: (1) binding over and around neuronal cell bodies, (2) receptors locally surrounding neurons, (3) dense punctate binding in the dorsal CN (DCN) not associated with neuronal cell bodies, and (4) diffuse fields of alpha Bgt receptors prominent in the DCN molecular layer, a field underlying the granule cell layer and in the medial sheet. The perikaryial receptors are abundant in the ventral CN (VCN) and are always associated with neurons expressing mRNA for the receptor. Other neurons in the VCN also express alpha 7 mRNA, but without alpha Bgt receptor expression associated with the cell body. In general, alpha Bgt receptor distribution parallels cholinergic terminal distribution, except in granule cell regions rich in cholinergic markers but low in alpha Bgt receptors. The findings indicate that alpha Bgt receptors are widespread in the CN but are selectively localized on somata, proximal dendrites, or distal dendrites depending on the specific CN subregion. The data are consistent with the hypothesis that descending cholinergic fibers modulate afferent auditory signals by regulating intracellular Ca2+ through alpha Bgt receptors.

Dopamine transporter development in postnatal rat striatum: an autoradiographic study with [3H]WIN 35,428.

The dopamine transporter mediates the reinforcing effects of cocaine, thus playing a central role in human cocaine addiction, and perhaps providing the mechanism for inducing the effects of prenatal cocaine exposure. This possibility has stimulated growing interest in the normal and abnormal development of this transporter. [3H]WIN 35,428 is a cocaine analog that is useful for studying the distribution and density of the dopamine transporter in striatum and other brain regions. The postnatal development of the dopamine transporter in the rat striatum was measured by quantitative autoradiography with [3H]WIN 35,428. Dopamine transporter levels were low at birth, increased through day 15, followed by much more rapid growth in late postnatal development. The majority of the transporter sites appeared after day 15. Lateral to medial and anterior to posterior gradients in transporter density were established early during development, and there was also an early concentration of transporter in striosomes that became difficult to identify by day 15. Differences between the developmental patterns described here and studies using other ligands for the dopamine transporter suggest there are significant differences in the transporter binding sites for these drugs. These differences in transporter ligand binding characteristics may reflect developmental changes in post-translational modification of the transporter and/or changes in the functional activity rather than simply the presence of the transporter.

Regional distribution of a creatine transporter in rat auditory brainstem: an in-situ hybridization study.

The expression of an mRNA encoding a creatine transporter (CRT1) was investigated in the rat auditory system under ambient sound conditions, using radiolabeled and non-radiolabeled oligonucleotide in-situ hybridization. The results indicated that CRT1 mRNA is widely distributed in auditory nuclei, including the fusiform and deep layers of the dorsal cochlear nucleus, the ventral cochlear nucleus, the superior olivary complex, the nuclei of the lateral lemniscus and the inferior colliculus. The molecular layer of the dorsal cochlear nucleus and the medial geniculate have low levels of label. Creatine provides cells with a reservoir of high-energy phosphate. Neurons do not synthesize creatine but accumulate it by a transport mechanism, which is probably the limiting step in the regulation of intracellular creatine. Therefore, the quantity of transporter expressed may reflect the utilization of creatine and could serve as an in-vitro indicator of endogenous high-energy metabolism in some cells. Although most auditory nuclei express CRT1 mRNA, the quantity of CRT1 mRNA varies among auditory nuclei, indicating that many auditory nuclei have high and fluctuating energy requirements. The level of CRT1 transcript or protein may be regulated by chronic metabolic changes in the auditory system that may occur, for example, with damage to the acoustic organ or the aging process.

Postnatal development of the dopamine transporter: a quantitative autoradiographic study.

The dopamine transporter performs an important role in regulating neurochemical transmission at dopaminergic synapses, as well as dopamine synthetic activity in dopaminergic neurons. Certain drugs and toxins exert effects at the transporter, especially cocaine, a common drug of abuse. We studied the development of these sites in the rat at postnatal ages day 0, 5, 10, 15, 21 and adult using quantitative autoradiography with the cocaine analogue [125I]RTI-55. At birth, certain structures such as the substantia nigra, interstitial nucleus of the medial longitudinal fasciculus, frontal and parietal cortex, and substantia inominata had [125I]RTI-55 binding levels that were already near the adult value. The striatum developed later, showing earlier growth in the anterior and dorsolateral regions, with early localization in both striosomes and a subcallosal streak. Anterior-to-posterior and lateral-to-medial gradients were present at day 0. The anterior striatum, ventral tegmental region, substantia nigra compacta and bed nucleus of the stria terminal is showed transient peaks in binding levels that were higher than the adult values. Structures showing relatively late development included the prefrontal cortex, nucleus accumbens shell, olfactory tubercle and subthalamic nucleus. Knowledge of the differential developmental patterns of the dopamine transporter in different brain regions may have implications for understanding the neurodevelopmental effects of prenatal cocaine exposure.

6-Hydroxydopamine-mediated induction of rat brain metallothionein I mRNA.

Oxidative stress, resulting either from excess generation or reduced scavenging of free radicals, has been proposed to play a role in damaging striatal neurons in Parkinson's disease. Since metallothionein is able to regulate the intracellular redox potential, we have undertaken a group of experiments to learn whether or not 6-hydroxydopamine, which generates free radicals and is toxic to dopaminergic neurons, could alter the levels of zinc and metallothionein in the brain. The lesioning of the rat striatum with 6-hydroxydopamine (8.0 micrograms in 4 microliter 0.02% ascorbic acid) resulted in a reduction in the levels of zinc and metallothionein in the striatum but not other brain regions tested. However, the intracerebroventricular administration of 6-hydroxydopamine, in a dosage regimen that does not lesion catecholaminergic pathways but causes oxidative stress, enhanced dramatically the level of metallothionein I mRNA in some brain areas such as hippocampus, arcuate nucleus, choroid plexus, and granular layer of cerebellum, but not in the caudate putamen. The results of these studies are interpreted to suggest that zinc or metallothionein are altered in conditions where oxidative stress has taken place. Moreover, it is proposed that areas of brain, such as striatum containing high concentrations of iron, but low levels of inducible metallothionein are particularly vulnerable to oxidative stress.

17 beta-Estradiol inhibits the production of dopamine by the tuberoinfundibular dopaminergic neurons of the male rat.

Previous work in our laboratory suggests that the indirect stimulation of prolactin gene transcription observed in male rats between 3 and 6 h following estrogen injection results from an inhibition of dopamine release from the tuberoinfundibular dopaminergic neurons. To evaluate further the role of dopamine in the regulation of prolactin gene transcription by estrogens, we have examined the acute effects of 17 beta-estradiol (E2) on dihydroxyphenylalanine accumulation in the arcuate median eminence region and the amount of dopamine associated with the arcuate median eminence region. Dihydroxyphenylalanine accumulation in the arcuate median eminence region was unaffected when examined 2 h following a single injection of E2 (10 micrograms), and was reduced by 60% when examined 4 h following E2 treatment. When examined 4 h after E2 injection, dopamine content in the arcuate median eminence region was significantly increased. These data suggest that estrogens exert acute inhibitory effects on the tuberoinfundibular dopaminergic neurons of the male rat, and further support the assertion that the indirect stimulatory actions of estrogens on prolactin gene transcription result from inhibition of hypothalamic dopamine production.

Localization and quantification of the dopamine transporter: comparison of [3H]WIN 35,428 and [125I]RTI-55.

Transport into the presynaptic terminal by the dopamine transporter is the primary mechanism for removing dopamine from the synaptic cleft. This transporter is a specific marker for dopamine terminals and is a primary site for CNS actions of cocaine. Several radioligands have been developed for analysis of the dopamine transporter. The ligands vary in affinity and specificity, leading to differences in reported transporter density in brain regions. We compared two of the most commonly used ligands, [3H]WIN 35,428 and [125I]RTI-55, analyzing the localization and density of sites in the rat brain using serial sections and quantitative autoradiography. Citalopram at 50 nmol/l was used to block [125I]RTI-55 binding to serotonin transport sites. Transporter density was highest in the striatum and both ligands labeled equivalent numbers of sites, with lateral to medial and anterior to posterior gradients. In most areas the density of sites measured with the two ligands was similar. However, [125I]RTI-55 binding was significantly higher than [3H]WIN 35,428 binding in the substantia nigra zona compacta, ventral tegmental area, subthalamic nucleus and a number of other subcortical nuclear groups while [3H]WIN 35,428 binding was higher in lateral striatum and in olfactory tubercle. These differences could reflect different forms of the transporter, perhaps due to post-translational modifications, and they may provide a basis for differential pharmacological regulation of transporter function in discrete brain regions and disease states.

Expression and regulation of brain metallothionein.

Many, but not all, zinc-containing neurons in the brain are a subclass of the glutamatergic neurons, and they are found predominantly in the telencephalon. These neurons store zinc in their presynaptic terminals and release it by a calcium-dependent mechanism. These "vesicular" pools of zinc are viewed as endogenous modulators of ligand- and voltage-gated ion channels. Metallothioneins (MTs) are low molecular weight zinc-binding proteins consisting of 25-30% cysteine, with no aromatic amino acids or disulfide bonds. The areas of the brain containing high contents of zinc such as the retina, the pineal gland, and the hippocampus synthesize unique isoforms of MT on a continuous basis. The four MT isoforms are thought to provide the neurons and glial elements with mechanisms to distribute, donate, and sequester zinc at presynaptic terminals; or buffer the excess zinc at synaptic junctions. In this cause, glutathione disulfide may participate in releasing zinc from MT. A similar nucleotide and amino acid sequence has made it difficult to obtain cDNA probes and antibodies capable of distinguishing indisputably among MT isoforms. MT-I and MT-II isoforms are found in the brain and in the peripheral tissues; MT-III isoform, possessing an additional seven amino acids, is expressed mostly in the brain and to a very minute extent in the intestine and pancreas; whereas MT-IV isoform is found in tissues containing stratified squamous epithelial cells. Since MTs are expressed in neurons that sequester zinc in their synaptic vesicles, the regulation of the expression of MT isoforms is extremely important in terms of maintaining the steady-state level of zinc and controlling redox potentials. The concentration of zinc has been shown to be altered in an extensive number of disorders of the central nervous system, including alcoholism. Alzheimer-type dementia, amyotrophic lateral sclerosis, Down's syndrome, epilepsy, Friedreich's ataxia, Guillaine-Barré syndrome, hepatic encephalopathy, multiple sclerosis, Parkinson's disease, Pick's disease, retinitis pigmentosa, retinal dystrophy, schizophrenia, and Wernicke-Korsakoff syndrome. The status of MT isoforms and other low molecular weight zinc-binding proteins in these conditions, diseases, disorders, or syndromes is being delineated at this time. Since several of these disorders, such as amyotrophic lateral sclerosis, are associated with oxidative stress, and since MT is able to prevent the formation of free radicals, it is believed that cytokine-induced induction of MT provides a long-lasting protection to avert oxidative damage.

In situ hybridization analysis of CHOT1, a creatine transporter, in the rat central nervous system.

A putative choline transporter (CHOT1) has been cloned from rat brain and is reported to express a high-affinity, sodium-dependent, hemicholinium-3-insensitive choline transporter in oocytes. A second transporter (OCCREATRA) cloned from rabbit brain is 98% homologous to CHOT1 and is reported to transport creatine. We examined the distribution of CHOT1 mRNA in rat brain by in situ hybridization, using a 48 base oligonucleotide probe. In adult rats, the hybridization signal was widespread, but with a distinct pattern. High levels of expression were detected in the cerebellum (Purkinje and granule cell layers), choroid plexus, medial habenula, pontine nuclei, several brainstem nuclei, and hippocampus (pyramidal cell layer). Moderate signal was detected in cortex, globus pallidus, corpus callosum, and most other white matter tracts. Very low levels were present in striatum, nucleus accumbens, hippocampus molecular layer, and cerebellar molecular layer. Emulsion autoradiography indicated cellular localization to both neurons and glia. CHOT1 mRNA was relatively abundant in some cholinergic regions, including the medial habenula, the medial septum, and several brainstem nuclei. However, the overall pattern was distinctly different from that expected for cholinergic markers and correlated well with the localization of creatine kinase. The widespread distribution and poor correlation with cholinergic markers indicates that the CHOT1 gene does not encode the classical choline transporter known to be associated with acetylcholine synthesis. It is possible that CHOT1 is associated with cholinergic neurotransmission in some brain regions. However, it appears to encode that the rat creatine transporter, and its widespread and heterogeneous distribution suggests regions where creatine phosphate is an important energy source.

Localization of nicotinic cholinergic receptors in rat brain: autoradiographic studies with [3H]cytisine.

There is a great deal of interest in the role of nicotinic acetylcholine receptors in the central nervous system, although their function is not well understood at present. Currently, central nicotinic receptors can be classified broadly as either alpha-bungarotoxin binding sites with low affinity for acetylcholine agonists, or as high-affinity agonist binding sites with low affinity for alpha-bungarotoxin. Neuronal nicotinic receptors with a high affinity for agonists are distributed widely in the central nervous system. Evidence from molecular biology and electrophysiology suggests that multiple nicotinic receptor types exist in the brain. In this study we have used the agonist [3H]cytisine as a ligand for autoradiography to generate a detailed quantitative map of the high-affinity agonist binding nicotinic receptor in the rat brain. Optimized binding conditions, characterization of the kinetic and equilibrium binding properties, and demonstration of the nicotinic pharmacology of this binding site in tissue sections confirm the usefulness of [3H]Cytisine as a ligand for nicotinic receptor autoradiography. [3H]Cytisine autoradiography provides excellent anatomic resolution with very low non-specific binding. This property has allowed us to describe variations in receptor density within subnuclei and gradients of receptor density in larger brain regions. Data from several studies suggest that the predominant high-affinity agonist binding nicotine receptor in the central nervous system is composed of the alpha 4 and beta 2 subunits. The data in the current study are consistent with the suggestion that [3H]cytisine labels only the alpha 4 beta 2 nicotinic receptor with high affinity, offering the possibility of localizing a specific nicotinic receptor subtype in the central nervous system. In summary, we characterize the optimum experimental conditions for the use of [3H]cytisine in tissue section autoradiography. [3H]Cytisine proves to be an excellent marker for nicotinic cholinergic receptors with a very high affinity and very low background. We provide a detailed quantitative characterization of nicotinic receptor density in the rat central nervous system and we find there are significant variations and gradients in receptor density within specific brain regions, including subregions previously thought to be homogeneous.