Ron S. Broide

Nicotine and brain development

Preclinical studies, using primarily rodent models, have shown acetylcholine to have a critical role in brain maturation via activation of nicotinic acetylcholine receptors (nAChRs), a structurally diverse family of ligand-gated ion channels. nAChRs are widely expressed in fetal central nervous system, with transient upregulation in numerous brain regions during critical developmental periods. Activation of nAChRs can have varied developmental influences that are dependent on the pharmacologic properties and localization of the receptor. These include regulation of transmitter release, gene expression, neurite outgrowth, cell survival, and synapse formation and maturation. Aberrant exposure of fetal and neonatal brain to nicotine, through maternal smoking or nicotine replacement therapy (NRT), has been shown to have detrimental effects on cholinergic modulation of brain development. These include alterations in sexual differentiation of the brain, and in cell survival and synaptogenesis. Long-term alterations in the functional status and pharmacologic properties of nAChRs may also occur, which result in modifications of specific neural circuitry such as the brainstem cardiorespiratory network and sensory thalamocortical gating. Such alterations in brain structure and function may contribute to clinically characterized deficits that result from maternal smoking, such as sudden infant death syndrome and auditory-cognitive dysfunction. Although not the only constituent of tobacco smoke, there is now abundant evidence that nicotine is a neural teratogen. Thus, alternatives to NRT should be sought as tobacco cessation treatments in pregnant women.

Developmental expression ofα7 neuronal nicotinic receptor messenger RNA in rat sensory cortex and thalamus

The distribution of alpha 7 messenger RNA expression was characterized in developing rat cortex and thalamus. Northern blot analysis of neonatal and adult cortex revealed a single messenger RNA transcript of 5.7 kb. Using in situ hybridization with both full length and short 35S-labeled alpha 7 riboprobes, a distinct transient expression of messenger RNA within sensory cortex and thalamus, during early postnatal development, was observed. alpha 7 transcripts were expressed in low levels as early as embryonic day 13 in the ventricular zone of the neocortex, and as early as embryonic day 15 in the thalamic neuroepithelium. A marked increase in messenger RNA levels was observed during the late prenatal period in both sensory and non-sensory regions of the cortex and thalamus. Moderate to high levels of messenger RNA were maintained into the first postnatal week, followed by a decline into adulthood. alpha 7 messenger RNA expression was significantly higher in the anterodorsal, lateral dorsal, ventral posterior medial and ventral posterior lateral thalamic nuclei of postnatal day 7 pups than in adult brains. Expression of messenger RNA within dorsal lateral geniculate, ventral lateral geniculate and medial geniculate did not show a significant reduction with age. Within the developing cortex, messenger RNA expression delineated the primary somatosensory, auditory and visual cortices in a unique laminar pattern that was consistently and significantly higher than in the adult in superficial layer VI. Higher levels of expression were also observed in retrosplenial cortex at postnatal day 7 than in the adult. Tangential sections through postnatal day 7 cortex revealed low levels of alpha 7 messenger RNA expression delineating the primary sensory areas in layer IV, corresponding to acetylcholinesterase-labeled thalamocortical afferents. However, these sensory areas exhibited higher levels of alpha 7 messenger RNA expression and were more clearly defined in layer VI, but not by acetylcholinesterase staining. The distribution of alpha 7 messenger RNA within the developing thalamocortical system parallels the distribution of alpha-bungarotoxin binding sites and suggests that the receptor is localized on both thalamic cells and their cortical target neurons. This transient and distinct pattern of distribution of the alpha 7 neuronal nicotinic receptor, which coincides with the major phase of thalamocortical development, suggests that it may play a functional role in the development of cortical circuitry.

Mice Homozygous for the L250T Mutation in the α7 Nicotinic Acetylcholine Receptor Show Increased Neuronal Apoptosis and Die Within 1 Day of Birth

Abstract: The α7 nicotinic acetylcholine receptor (nAChR) has been implicated in modulating neurotransmitter release and may play a role in the regulation of neuronal growth and differentiation. A threonine for leucine 247 substitution in the channel domain of the chick α7 nAChR increases agonist affinity and decreases the rate of desensitization, creating a “gain of function” model for this receptor. We have generated mice that express the analogous mutation (L250T) in the α7 nAChR using the techniques of homologous recombination and here report their characteristics. Mice heterozygous (+/T) for the L250T mutation are viable, fertile, and anatomically normal compared with wild‐type littermates. In contrast, homozygous (T/T) L250T mice die within 2‐24 h of birth. Brains of T/T mouse pups exhibit a marked reduction in α7 nAChR protein levels and show extensive apoptotic cell death throughout the somatosensory cortex. Furthermore, α7 L250T nAChRs are functionally expressed on neurons within the brains of T/T neonatal mice and have properties that are consistent with those observed for the rat α7 L250T and the chick α7 L247T mutant nAChRs expressed in oocytes. These findings indicate that neurons in the developing brain expressing only α7 L250T mutant nAChRs are susceptible to abnormal apoptosis, possibly due to increased Ca2+ influx.

Expression of α2 adrenoceptors during rat brain development—I. α2A messenger RNA expression

The distribution of alpha 2A adrenoceptor messenger RNA expression in developing rat brain was characterized using in situ hybridization with 35S-labeled riboprobes. Intense hybridization signal was detected as early as embryonic day 14 in several areas adjacent to the forebrain and hindbrain germinal zones and in central noradrenergic neurons. A marked increase in messenger RNA expression was observed throughout the brain during late prenatal development, consistent with the migration and maturation of neurons in developing brain structures. In embryonic brain, there was a temporal and spatial correspondence in the appearance of alpha 2A messenger RNA expression and binding sites labeled with [3H]idazoxan or p-[125I]iodoclonidine, indicating translation into receptor protein at an early stage of development. Whereas the presynaptic expression remained constant throughout development, there was an early postnatal decline of alpha 2A receptor expression in many brain regions, including the olfactory bulb, cortex, caudate-putamen, hippocampus, thalamus, hypothalamus and medulla. Thereafter, messenger RNA expression increased, establishing an adult-like pattern during the second postnatal week, but remained low in areas such as the caudate-putamen, thalamus and hippocampus, which do not exhibit extensive expression in the adult. The transient perinatal expression of this alpha 2 adrenoceptor type, which coincides with a period of hyperreactivity to sensory stimuli in the locus coeruleus, may indicate a specific functional role for the alpha 2A receptor in the developing rat brain. The early and intense expression in olfactory structures suggests an involvement in early olfactory learning. The pattern of widespread, transient expression of alpha 2A receptors in the fetal brain is in marked contrast to the postnatal development of the alpha 2C receptor type.

Expression of α2 adrenoceptors during rat brain development—II. α2C messenger RNA expression and [3H]rauwolscine binding

Abstract The distributions of α 2C adrenoceptor messenger RNA and high-affinity [ 3 H]rauwolscine binding sites were characterized in developing rat brain. Using in situ hybridization with 35 S-labeled riboprobes directed against the third intracellular loop, α 2C messenger RNA expression appeared in an adult-like pattern during the first and second postnatal weeks, in the anterior olfactory nucleus, caudate–putamen, olfactory tubercles, islands of Calleja and hippocampus, following the time-course of maturation of these structures. Only in the cerebellum was α 2C messenger RNA transiently expressed during the critical period of granule cell development. High-affinity [ 3 H]rauwolscine binding sites were detected using receptor autoradiography and revealed a similar spatial and temporal time-course of appearance during rat brain development. The highest numbers of binding sites were detected in the olfactory tubercles and islands of Calleja, and moderate numbers in the anterior olfactory nucleus, caudate–putamen and hippocampus. Like α 2C messenger RNA expression, high-affinity [ 3 H]rauwolscine binding sites were transiently expressed in the cerebellum. In some areas (e.g., the substantia nigra), [ 3 H]rauwolscine binding sites were detected even though α C2 messenger RNA expression was absent. The strong spatial and temporal correspondence between messenger RNA expression and radioligand binding supports the conclusion that [ 3 H]rauwolscine selectively labels α 2C adrenoceptors in the rat brain. The developmental pattern which was observed is in marked contrast to the early, transient expression of the α 2A adrenoceptor. Thus, the α 2A and α 2C receptor types may serve distinct functional roles in the developing brain.

Highly sensitive radioactive in situ hybridization using full length hydrolyzed riboprobes to detect α2 adrenoceptor subtype mRNAs in adult and developing rat brain

The mRNA expression of highly homologous alpha 2 adrenoceptor subtypes was determined using a highly sensitive in situ hybridization protocol that allowed the detection of low abundance mRNA. Full-length 35S-labeled riboprobes specific for alpha 2A, alpha 2B and alpha 2C adrenoceptors were used for maximal sensitivity. The probes were hydrolyzed to an average length of 600 bp which, in combination with proteinase K digestion, resulted in optimal probe penetration in developmental and adult tissue. The expression intensity could be quantified and the ontogeny of receptor mRNA expression determined. At the same time receptor binding sites or functional proteins could be detected simultaneously in adjacent sections, because fresh frozen and post-fixed tissue was used.

Calcium Regulation of Agonist Binding to α7‐Type Nicotinic Acetylcholine Receptors in Adult and Fetal Rat Hippocampus

Abstract: Quantitative autoradiography was used to compare the binding properties of α7‐type nicotinic acetylcholine receptors in fetal and adult rat hippocampus. Whereas there were high levels of 125I‐α‐bungarotoxin (125I‐α‐BTX) binding throughout fetal hippocampal field CA1, there was a significant decrease in binding site density in the adult. The affinity of 125I‐α‐BTX binding, as well as α‐cobratoxin and nicotine potency to displace 125I‐α‐BTX, did not change with age. Addition of Ca2+ to the assay buffer did not alter 125I‐α‐BTX binding, or α‐cobratoxin inhibition of 125I‐α‐BTX binding, although it significantly increased nicotine affinity at both ages. The effect of Ca2+ on agonist affinity was dose‐dependent, with an EC50 value of 0.25–0.5 mM. Ca2+ also significantly increased the cooperativity of nicotine displacement curves in stratum oriens of the adult, but not in the fetus. These findings indicate that the properties of hippocampal 125I‐α‐BTX binding sites are largely similar across age. Ca2+ selectively enhances the affinity of agonist binding, with no change in antagonist binding. This ionic effect may result from potentiation of agonist binding to a desensitized state of the α7 nicotinic acetylcholine receptor and may represent an important neuroprotective mechanism.

Possible Cholinergic and Non-Cholinergic Actions of Transiently Expressed Acetylcholinesterase in Thalamocortical Development Projections

Recent studies from this laboratory and others have demonstrated that acetylcholinesterase (AChE) activity is expressed transiently in developing cerebral cortex of mammals (Kostovic and Goldman-Rakic, 1983; Kostovic and Rakic, 1984; Kristt and Waldman, 1981; Robertson, 1987; Robertson et al., 1988; Robertson and Yu, 1994; Schlaggar and O’Leary, 1994). Studies in rodents have demonstrated that the transiently expressed AChE is produced by the protein synthetic apparatus in the perikaryal cytoplasm of thalamic neurons and is transported along thalamocortical axons to their terminals in primary sensory cortex (Robertson, 1991; Robertson et al., 1988; Robertson and Yu, 1994). The AChE activity can first be detected in rats around the time of birth, reaches peak intensity during the second postnatal week, and then subsides to adult levels during the third postnatal week (Robertson, 1987). Interestingly, this temporal pattern of AChE expression coincides closely with the timing of thalamocortical axonal ingrowth into cerebral cortex (Catalano et al., 1991; Kageyama and Robertson, 1993; Schlaggar and O’Leary, 1994).