L. Charles Murrin

Nicotine administration to rats: methodological considerations.

The effects of nicotine on normal physiological function are of increasing concern. Preliminary to studies on the effects of prenatal exposure to nicotine, we examined methods of administering nicotine to rats. Drinking water containing nicotine was not palatable to rats and was an unsatisfactory method in our hands, producing weight loss and large decreases in fluid intake. Administration of nicotine in a complete liquid diet produced better results but the data suggest that oral administration of nicotine may interfere with absorption of some nutrients. Osmotic mini-pumps were found to be the best mechanism of nicotine delivery of those tried. There were no significant effects on food or water intake nor on weight gain, particularly when using a short term anesthetic for pump implantation. Plasma nicotine and cotinine levels were directly correlated to dose of nicotine delivered. Plasma nicotine levels similar to levels reported in humans were obtained.

High‐Affinity Choline Transport Sites: Use of [3H]Hemicholinium‐3 as a Quantitative Marker

Abstract: High‐affinity choline transport (HAChT), the rate‐limiting and regulatory step in acetylcholine (ACh) synthesis, is selectively localized to cholinergic neurons. Hemicholinium‐3 (HC3), a potent and selective inhibitor of HAChT, has been used as a specific radioligand to quantify HAChT sites in membrane binding and autoradiographic studies. Because both HAChT velocity and [3H]HC3 binding change as in vivo activity of cholinergic neurons is altered, these markers are also useful measures of cholinergic neuronal activity. Evidence that [3H]HC3 is a specific ligand for HAChT sites on cholinergic terminals is reviewed. The ion requirements of HAChT and [3H]HC3 binding indicate that sodium and chloride are required for recognition of both choline and [3H]HC3. A common recognition site is also indicated by the close correspondence of the potency of HC3 and choline analogues for inhibiting both HAChT and [3H]HC3 binding. The parallel regional distributions of both markers in adult brain, during development and after specific lesions, all indicate specific cholinergic localization. The close association of HAChT and [3H]HC3 binding sites is also supported by parallel regulatory changes occurring after in vivo drug treatments and in vitro depolarization. Overall, the data indicate a close association between HAChT and [3H]HC3 binding and are consistent with the sites being identical. Methodologic considerations in using [3H]HC3 as a ligand and considerations in interpretation of results are also discussed.

Dopamine D1 receptor development in the rat striatum: early localization in striosomes ☆

The development of dopamine D1-receptors in rat striatum during the early postnatal period is examined, using autoradiography and [3H]SCH 23390 as ligand. Dopamine D1-receptors are present in striatum at birth and are more dense than in any adjacent region. The receptors are preferentially localized in striosomes and to some extent in a subcallosal streak. The density of D1-receptors in the matrix increases with age so that by two weeks postnatally the striosomal pattern is no longer evident, and the overall dense labelling is the same as seen in adults. Dopamine D1-receptor development seems to take place earlier than that of dopamine terminals but at the same time as or somewhat later than that of acetylcholinesterase. The D1-defined striosomes move from ventrolateral towards dorso-medial striatum with increasing age and from anterior to posterior striatum. This direction is nearly perpendicular to the direction of development of several other markers, including dopamine terminals and D2-receptors. The present studies indicate that for markers appearing in the striosomal compartment there are different patterns of development with respect to time and spatial pattern. Regulation of striatal development by interaction of neuronal systems with one another and with other factors is complex and will require extensive study to clarify the mechanisms involved.

Postnatal development of the dopamine transporter: a quantitative autoradiographic study

The dopamine transporter performs an important role in regulatinf neurochemical transmission at dopaminergic synapses, as well as dopamine synthetic activity in dopaminergic neurons. Certain drugs and toxins exert effects at the transportted, especially cocaine, a common drug of abuse. We studied the development of these sites in the rat 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 he 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. Anterio-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 terminals 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.

Postnatal ontogeny of dopamine D2 receptors in rat striatum

Studies of the ontogeny of dopamine D2 receptors in rat striatum were carried out using [3H]spiroperidol as ligand in the presence of 30 nM ketanserin. D2 receptors increased with age, and saturation studies indicated that this was due to an increase in receptor density (Bmax) and not to a change in affinity (KA). By 21 days of age, receptor density had reached adult levels. Hill plots indicated no cooperativity in the binding from 7 to 21 days of age. Pharmacologically, receptors at 7, 14 and 21 days of age were similar to those of adult rats, exhibiting characteristics of dopamine D2 receptors. These studies provide a basic analysis of dopamine D2 receptors in rat striatum during early postnatal development.

Ontogeny of dopamine, serotonin and spirodecanone receptors in rat forebrain — An autoradiographic study

Sections from freshly frozen neonatal rat brain, ages 0-21 days, were incubated with [3H]spiperone (SP). Initial studies characterized the binding sites for SP in terms of association and dissociation rates, saturability and pharmacology. The binding sites were found to be predominantly dopamine D2 receptors in sections centered in the striatum and these receptors were similar to receptors in adult brain. Autoradiographic studies using in vitro techniques examined the anatomic distribution of [3H]SP binding sites. Using domperidone, ketanserin and (+)butaclamol it was possible to differentiate dopamine D2, serotonin S2 and spirodecanone receptors. Dopamine receptors were found at birth in striatum and nucleus accumbens and increased in density with age. In the first two weeks postnatally there was an apparent dorsolateral to ventromedial gradient in the striatum. Serotonin S2 receptors were found in the cortex, lateral olfactory tubercles and nucleus accumbens and claustrum. These receptors increased in density with age but to a much lesser extent than dopamine receptors. Spirodecanone receptors were first apparent in the piriform cortex by day 5. They became very dense with age in particular regions, i.e., layer II of cortex, piriform cortex, medial olfactory tubercles, lateral septum and in patches in the nucleus accumbens. These studies delineate with a high resolution at an anatomical level the major receptor sites for neuroleptic drugs in the developing rat forebrain. They describe the developmental pattern of these receptors and provide a basis for further studies on their control and function during development.

Ontogeny of the rat striatum: Correspondence of dopamine terminals, opiate receptors and acetylcholinesterase

The ontogeny of dopamine terminals, mu opiate receptors and acetylcholinesterase (AChE) in rat striatum were examined during the first two weeks postnatally using fluorescence histochemistry, autoradiography and AChE stain, respectively. The most dense regions of all three markers were superimposable, particularly in the dorsal third of the striatum, as long as the dense regions could be clearly visualized. This anatomic overlapping of markers for three neuronal systems suggests an important and distinct relationship between these neuronal systems during development in addition to that already seen in adult tissue.

Ontogeny of dopamine D1 receptors in rat forebrain: a quantitative autoradiographic study

The development of dopamine D1 receptors during the early postnatal period is examined in rat forebrain, using quantitative autoradiography and [3H]SCH 23390 as ligand. Dopamine D1 receptors are present in many regions at birth. In general, regions with the highest densities of D1 receptors in adults have the highest densities of receptors in neonates. For most regions in the forebrain there is a steady increase in the density of D1 receptors, as measured in fmol/mg tissue, from day 1 to day 28 of age. This is most obvious in the regions with the greatest number of receptors, such as the caudate-putamen, the nucleus accumbens, and the olfactory tubercles. The more caudal regions examined in this study had a relatively greater portion of their receptors present at day 1 compared to day 28 than more rostral regions. In general they had about 50% of their receptors present at birth, whereas most regions studied had receptor levels at day 1 about 20% of those found at day 28. In the most anterior regions, the development of receptors was somewhat slower. Receptor number in the frontal cortex region did not begin to increase until about 10 days of age. The present studies indicate that dopamine D1 receptors develop in the forebrain of the rat in a steady pattern. There are no dramatic increases or decreases in receptor number throughout the postnatal period.

Ontogeny of Dopamine D1 Receptors in Rat Striatum

The development of dopamine D1 receptors in rat striatum during the early postnatal period is described, using [3H]piflutixol as ligand. Dopamine D1 receptors increase in number from day of birth until about 21 days of age, when they reach adult levels. This increase in number parallels the increase in several other dopamine markers in striatum during the same time period. The increase is reflected in an increase in Bmax of ligand binding to D1 receptors. All other properties of D1 receptors that were examined do not change throughout this developmental period and are essentially the same as those found in adult tissue. These include association and dissociation rates, affinity for piflutixol as determined by kinetic and saturation studies, and pharmacology. These studies provide a biochemical and pharmacological basis for further studies on the ontogeny of dopamine receptors and of striatum and on factors regulating development of this region.

Mitochondrial fragmentation is involved in methamphetamine-induced cell death in rat hippocampal neural progenitor cells.

Methamphetamine (METH) induces neurodegeneration through damage and apoptosis of dopaminergic nerve terminals and striatal cells, presumably via cross-talk between the endoplasmic reticulum and mitochondria-dependent death cascades. However, the effects of METH on neural progenitor cells (NPC), an important reservoir for replacing neurons and glia during development and injury, remain elusive. Using a rat hippocampal NPC (rhNPC) culture, we characterized the METH-induced mitochondrial fragmentation, apoptosis, and its related signaling mechanism through immunocytochemistry, flow cytometry, and Western blotting. We observed that METH induced rhNPC mitochondrial fragmentation, apoptosis, and inhibited cell proliferation. The mitochondrial fission protein dynamin-related protein 1 (Drp1) and reactive oxygen species (ROS), but not calcium (Ca2+) influx, were involved in the regulation of METH-induced mitochondrial fragmentation. Furthermore, our results indicated that dysregulation of ROS contributed to the oligomerization and translocation of Drp1, resulting in mitochondrial fragmentation in rhNPC. Taken together, our data demonstrate that METH-mediated ROS generation results in the dysregulation of Drp1, which leads to mitochondrial fragmentation and subsequent apoptosis in rhNPC. This provides a potential mechanism for METH-related neurodegenerative disorders, and also provides insight into therapeutic strategies for the neurodegenerative effects of METH.