Neuron specific alpha-adrenergic receptor expression in human cerebellum: implications for emerging cerebellar roles in neurologic disease.

Recent data suggest novel functional roles for cerebellar involvement in a number of neurologic diseases. Function of cerebellar neurons is known to be modulated by norepinephrine and adrenergic receptors. The distribution of adrenergic receptor subtypes has been described in experimental animals, but corroboration of such studies in the human cerebellum, necessary for drug treatment, is still lacking. In the present work we studied cell-specific localizations of alpha1 adrenergic receptor subtype mRNA (alpha 1a, alpha 1b, alpha 1d), and alpha2 adrenergic receptor subtype mRNA (alpha 2a, alpha 2b, alpha 2c) by in situ hybridization on cryostat sections of human cerebellum (cortical layers and dentate nucleus). We observed unique neuron-specific alpha1 adrenergic receptor and alpha2 adrenergic receptor subtype distribution in human cerebellum. The cerebellar cortex expresses mRNA encoding all six alpha adrenergic receptor subtypes, whereas dentate nucleus neurons express all subtype mRNAs, except alpha 2a adrenergic receptor mRNA. All Purkinje cells label strongly for alpha 2a and alpha 2b adrenergic receptor mRNA. Additionally, Purkinje cells of the anterior lobe vermis (lobules I to V) and uvula/tonsil (lobules IX/HIX) express alpha 1a and alpha 2c subtypes, and Purkinje cells in the ansiform lobule (lobule HVII) and uvula/tonsil express alpha 1b and alpha 2c adrenergic receptor subtypes. Basket cells show a strong signal for alpha 1a, moderate signal for alpha 2a and light label for alpha 2b adrenergic receptor mRNA. In stellate cells, besides a strong label of alpha 2a adrenergic receptor mRNA in all and moderate label of alpha 2b message in select stellate cells, the inner stellate cells are also moderately positive for alpha 1b adrenergic receptor mRNA. Granule and Golgi cells express high levels of alpha 2a and alpha 2b adrenergic receptor mRNAs. These data contribute new information regarding specific location of adrenergic receptor subtypes in human cerebellar neurons. We discuss our observations in terms of possible modulatory roles of adrenergic receptor subtypes in cerebellar neurons responding to sensory and autonomic input signals, and review species differences in cerebellar adrenergic receptor expression.

Ontogeny of cholinergic neurons in the mouse forebrain.

The development of cholinergic neurons in the mouse forebrain was studied by immunocytochemistry with a monoclonal antibody to choline acetyltransferase (ChAT), the rate-limiting enzyme for acetylcholine synthesis. Since this antibody stained dividing cells in ventricular germinal zones as well as differentiating neurons, likely routes of migration could be inferred on the basis of the location of immunoreactive (IR) cells at different gestational ages. Germinal zones for cholinergic cells were observed in all ventricular zones of the forebrain with the ventral zones generating the earliest cells by gestational day 13.5 (GD13.5). On GD14, ChAT IR cells were visible in the germinal zones of the eye, olfactory ventricle, anterior horn, and dorsolateral aspect of the lateral ventricle, lateral ganglionic eminence, ventro- and dorsolateral third ventricle, and in the pineal anlage (epiphysis). ChAT IR neurons continued to develop in these and additional germinal zones on GD15, including the medial, dorsal, and dorsomedial walls of the lateral ventricle, and the medial and dorsal ganglionic eminence. On GD16, ChAT IR neurons were located in the prelimbic, pyriform, and parietal cortices and the lamina terminalis, and a cluster of IR cells was observed in the ventricular zone of the caudatopallial angle. On GD17-18, neurons in the anterior olfactory nucleus, olfactory tubercle, horizontal and vertical nucleus of the diagonal band, and medial septal nucleus stained more darkly and were multipolar, whereas immature bipolar neurons appeared to continue their migration into the hippocampus and along major fiber tracts, such as the corpus callosum, external capsule, fornix and anterior commissure. This study provides a comprehensive view of the zones of origin, probable routes of migration, and final destination of cholinergic neurons in the mouse forebrain.

Ontogeny of D1A and D2 dopamine receptor subtypes in rat brain using in situ hybridization and receptor binding.

The prenatal and postnatal ontogeny of D1A and D2 dopamine receptors was assessed by in situ hybridization of messenger RNAs encoding the receptors and by radioligand binding autoradiography. On gestational day 14, signals for D1A and D2 dopamine receptor messages were observed in selected regions in ventricular and subventricular zones which contain dividing neuroblasts, and in intermediate zones that contain maturing and migrating neurons. Specifically, D1A and D2 dopamine receptor message was observed in the developing caudate-putamen, olfactory tubercle, and frontal, cingulate, parietal and insular cortices. Additionally, D1A dopamine receptor messenger RNA was found in the developing epithalamus, thalamus, hypothalamus, pons, spinal cord and neural retina; D2 dopamine receptor messenger RNA was also observed in the mesencephalic dopaminergic nuclear complex. Gene expression of D1A and D2 dopamine receptor subtypes in specific cells as they differentiate precedes dopamine innervation and implies that receptor expression is an intrinsic property of these neurons. The early expression of dopamine receptor messenger RNA suggests a regulatory role for these receptors in brain development. While the signal for both messages increased in the intermediate zones on gestational day 16, it decreased in the ventricular and subventricular zones, and was no longer apparent in these zones by gestational day 18. By gestational day 18, abundant D1A or D2 dopamine receptor messenger RNA was observed in cell groups similar in location to those observed in the adult brain. On gestational day 18, D1A dopamine receptor message was noted in the neural retina, anterior olfactory nucleus, the insular, prefrontal, frontal, cingulate, parietal and retrosplenial cortices, the olfactory tubercle, caudate-putamen, lateral habenula, dorsolateral geniculate nucleus, ventrolateral and mediolateral thalamic nuclei, and the suprachiasmatic and ventromedial nuclei of the hypothalamus. D2 dopamine receptor message was observed on gestational day 18 in the insular, prefrontal, frontal and cingulate cortices, the olfactory tubercle, caudate-putamen, ventral tegmental area, substantia nigra, and the intermediate lobe of the pituitary. At birth, expression of messenger RNA for both dopamine receptor subtypes in the striatum approximated that seen in mature rats. In contrast, D1A and D2 receptor binding, measured with [3H]SCH-23390 and [3H]raclopride, respectively, was low at birth and progressively increased to reach adult levels between days 14 and 21. The in situ hybridization data showing early prenatal expression of messenger RNA for the D1A and D2 dopamine receptors are consistent with the hypothesis that these receptors have a regulatory role in neuronal development.(ABSTRACT TRUNCATED AT 400 WORDS)

Morphogenetic roles of acetylcholine.

In the adult nervous system, neurotransmitters mediate cellular communication within neuronal circuits. In developing tissues and primitive organisms, neurotransmitters subserve growth regulatory and morphogenetic functions. Accumulated evidence suggests that acetylcholine, (ACh), released from growing axons, regulates growth, differentiation, and plasticity of developing central nervous system neurons. In addition to intrinsic cholinergic neurons, the cerebral cortex and hippocampus receive extensive innervation from cholinergic neurons in the basal forebrain, beginning prenatally and continuing throughout the period of active growth and synaptogenesis. Acute exposure to ethanol in early gestation (which prevents formation of basal forebrain cholinergic neurons) or neonatal lesioning of basal forebrain cholinergic neurons, significantly compromises cortical development and produces persistent impairment of cognitive functions. Neonatal visual deprivation alters developmental expression of muscarinic acetylcholine receptors (mAChR) in visual cortex, whereas local infusion of mAChR antagonists impairs plasticity of visual cortical neurons. These findings raise the possibility that exposure to environmental neurotoxins that affect cholinergic systems may seriously compromise brain development and have long-lasting morphologic, neurochemical, and functional consequences.

Alpha1-adrenergic receptors in human spinal cord: specific localized expression of mRNA encoding alpha1-adrenergic receptor subtypes at four distinct levels.

alpha1-Adrenergic receptors (alpha1ARs) are important in lower urinary tract syndromes such as benign prostatic hypertrophy and bladder irritability. Spinal cord alpha1ARs have been postulated to play a role in modulating these diseases, yet alpha1AR subtype (alpha1a, alpha1b, alpha1d) neuronal localization in human spinal cord has not been described. We therefore tested the hypothesis that alpha1AR subtype distribution varies according to specific spinal cord tract and level. In situ hybridization was performed to identify cell bodies containing alpha1AR subtype mRNA at four levels of human spinal cord (cervical enlargement, thoracic, lumbar, sacral). alpha1AR mRNA is present in ventral gray matter only (ventral>dorsal; sacral>lumbar=thoracic>cervical). Signaling cell bodies were detected in anterior horn motor neurons at all levels; dorsal nucleus of Clarke and intermediolateral columns in cervical enlargement, thoracic and lumbar spinal cord regions; and parasympathetic nucleus in sacral spinal cord. Although all three alpha1AR subtypes are present throughout human spinal cord, alpha1d mRNA predominates overall. If confirmed at a protein level, these findings may contribute to the development of new therapeutic strategies in the treatment of several human diseases.

alpha 2-Adrenergic receptors in human spinal cord: specific localized expression of mRNA encoding alpha 2-adrenergic receptor subtypes at four distinct levels.

alpha 2-Adrenergic receptor (AR) subtype mRNA (alpha 2a, alpha 2b, alpha 2c) neuronal localization in human spinal cord has not been described. We therefore performed in situ hybridization to identify cell bodies at four levels of human spinal cord (cervical, thoracic, lumbar, sacral) containing alpha 2AR subtype specific mRNA. alpha 2AR mRNA is present in gray matter only (ventral > dorsal; sacral > cervical > thoracic = lumbar). In addition to alpha 2AR mRNA in cell bodies in thoracic and lumbar intermediolateral (sympathetic) and sacral intermediate (parasympathetic) cell columns (lamina VII), all levels in dorsal horn laminae I, II, V, and ventral horn lamina IX, we demonstrate alpha 2AR mRNA in dorsal horn laminae III and IV, and dorsal nucleus of Clarke, where alpha 2ARs have not been described. Previously unreported heterogeneity in alpha 2AR subtype distribution (alpha 2a and alpha 2bAR mRNA present, alpha 2cAR mRNA virtually absent) is found at all sites of alpha 2AR mRNA expression in human spinal cord, including locations known to mediate effects of alpha 2AR agonist drugs on nociception, autonomic function and motor tone. Cervical spinal cord demonstrates a predominance of alpha 2a mRNA signal, while thoracic, lumbar, and sacral spinal cord demonstrate an increasing predominance of alpha 2bAR mRNA. If confirmed at a protein level, these findings have profound implications for therapeutic strategies in managing human pain.

Distribution of alpha 2-adrenergic receptor subtype gene expression in rat brain.

alpha 2-Adrenergic receptors in brain are important presynaptic modulators of central noradrenergic function (autoreceptors) and postsynaptic mediators of many of the widespread effects of catecholamines and related drugs. alpha 2-Adrenergic agonists are currently used as antihypertensives and preanesthetic agents, but new subtype-selective alpha 2-adrenoceptor agonists and antagonists have additional therapeutic application potential. Three genes encoding specific alpha 2-adrenoceptor subtypes (alpha 2A, alpha 2B, and alpha 2C) have been isolated and characterized. RNA blotting indicates that all three are expressed in rat brain. This study used in situ hybridization with 35S-labeled RNA probes to map the distribution of alpha 2-adrenoceptor subtype gene expression in rat brain. alpha 2A mRNA was most abundant in the locus coeruleus, but was also widely distributed in the brain stem, cerebral cortex, septum, hypothalamus, hippocampus and amygdala. alpha 2B mRNA was observed only in the thalamus. alpha 2C mRNA was mainly localized to the basal ganglia, olfactory tubercle, hippocampus, and cerebral cortex. These mRNA distributions largely agree with previous findings on the alpha 2-adrenoceptor distributions in the rat brain, but suggest that the localization patterns for each receptor subtype are unique. The expression of alpha 2A mRNA in noradrenergic neurons indicates that this subtype mediates presynaptic autoreceptor functions. Furthermore, the localization of alpha 2A mRNA in noradrenergic projection areas suggests that this receptor may also have an important role in mediating postsynaptic effects. The precise physiological and pharmacological roles of the alpha 2-adrenoceptor subtypes are still largely unknown, but it is expected that in situ hybridization coupled to various methods to identify the transmitter phenotypes of the subtype-expressing neurons will help to clarify these important issues in the near future.

Development of neurotransmitter systems in the mouse embryo following acute ethanol exposure: a histological and immunocytochemical study.

Acute maternal ethanol administration to C57B1/6J mice on gestational day 7 (GD7) results in facial and brain abnormalities similar to those reported in human fetal alcohol syndrome (FAS). Using this model, we assessed the damage to brain structures using histological methods and changes in developing neurotransmitter systems with immunocytochemistry. Cholinergic neurons in the forebrain were stained with a monoclonal antibody to choline acetyltransferase (ChAT). Catecholaminergic neurons in the midbrain and serotoninergic neurons in the hindbrain were stained with polyclonal antisera to tyrosine hydroxylase (TH) and serotonin (5-HT), respectively. Forebrain deficiencies, including loss of midline structures (olfactory bulbs, midline septation, medial septal area) and deficits in lateral and dorsal regions (neostriatum and cerebral cortex) were found in both GD14 embryos and GD18 fetuses. In severely affected offspring, complete loss of the septal region resulted in conjoined lateral ventricles and a reduction in the thickness of the ventricular zone surrounding the single ventricle, as well as a severe loss of ChAT neurons which would normally be located in this territory. However, no consistent changes were seen in the distribution or size of TH or 5-HT neuronal cell groups in the midbrain and hindbrain. These differences in effects on specific neurotransmitter systems reflect the fact that the forebrain is most severely affected by early ethanol administration, whereas the hindbrain is relatively spared. Such differential effects could produce an imbalance in developing neurotransmitter systems in the embryonic and fetal brain, which could explain some of the functional deficits observed in children with FAS.

In situ hybridization: identification of rare mRNAs in human tissues.

In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non-selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32P or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).

An atlas of the prenatal mouse brain: gestational day 14.

A prenatal atlas of the mouse brain is presently unavailable and is needed for studies of normal and abnormal development, using techniques including immunocytochemistry and in situ hybridization. This atlas will be especially useful for researchers studying transgenic and mutant mice. This collection of photomicrographs and corresponding drawings of Gestational Day (GD) 14 mouse brain sections is an excerpt from a larger atlas encompassing GD 12-18. In composing this atlas, available published studies on the developing rodent brain were consulted to aid in the detailed labeling of embryonic brain structures. C57Bl/6J mice were mated for 1 h, and the presence of a copulation plug was designated as GD 0. GD 14 embryos were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer and embedded in paraffin. Serial sections (10 microns thickness) were cut through whole heads in sagittal and horizontal planes. They were stained with hematoxylin and eosin and photographed. Magnifications were 43X and 31X for the horizontal and sagittal sections, respectively. Photographs were traced and line drawings prepared using an Adobe Illustrator on a Macintosh computer.

Prenatal expression of 5-HT1C and 5-HT2 receptors in the rat central nervous system.

The expression of mRNAs for two 5-HT receptors (5-HT1C, 5-HT2) has been investigated by evaluating in situ hybridization in the prenatal rat CNS. At Embryonic Day 14 (E14), the highest signal for 5-HT1C was found in the choroid plexus, while the marginal/intermediate (m/i) zones of the midbrain, brain stem (including monoaminergic groups), and spinal cord also displayed label. By E18-21 a number of more rostral regions contained transcript, including the hippocampus (CA1), in addition to more intense signal in midbrain, brain stem, and spinal cord. Expression in the choroid plexus appeared to peak between E16-E18, although considerable hybridization signal remained at E21. 5-HT2 transcripts were also detected at E14. Label was present in m/i zones of the midbrain and in a number of other areas. In comparison to 5-HT1C, 5-HT2 mRNA was distributed over a wider rostral-caudal extent at this age. As with 5-HT1C mRNA, signal increased over rostral and brain stem areas at late gestational ages with significant labeling appearing in the olfactory bulb, cerebellum, cortical plate and subplate, hippocampus (dentate gyrus), and monoaminergic nuclei. 5-HT1C and 5-HT2 receptor transcripts were also present over the meninges at E16 and may represent transient expression of these receptors. These expression patterns in the embryonic rat brain, in conjunction with previous evidence indicating that 5-HT can act as a differentiation signal for target neurons, suggests that prenatal 5-HT receptors are positioned to play a role in the prenatal development of the CNS.

D1 dopamine receptor binding and mRNA levels are not altered after neonatal 6-hydroxydopamine treatment: evidence against dopamine-mediated induction of D1 dopamine receptors during postnatal development.

The role of dopaminergic innervation on the postnatal developmental expression of D1 dopamine receptors was investigated. Bilateral destruction of dopamine-containing neurons was achieved by treating rats intracisternally with 6-hydroxydopamine (6-OHDA) on postnatal day 3, and rats were killed on day 21. To ensure effective reduction of D1 receptor activation by residual dopamine, a group of 6-OHDA-lesioned rats was given twice daily injections of the D1 receptor antagonist SCH-23390, from day 4 to 20. D1 dopamine receptor binding was assessed in the caudate-putamen, nucleus accumbens, and olfactory tubercle by quantitative autoradiographic analysis of [3H]SCH-23390 binding. In addition, the relative amount of D1A receptor mRNA was assessed by in situ hybridization of a 35S-labeled riboprobe. In the developing rats, neither the amount of [3H]SCH-23390 binding nor the amount of D1A receptor mRNA was altered by 6-OHDA lesioning followed by chronic treatment with SCH-23390. Thus, bilateral destruction of dopamine-containing neurons and treatment with SCH-23390 in neonatal rats did not interfere with the developmental expression of D1 receptors or alter the levels of mRNA that code for this receptor protein. Treatment of intact rats with SCH-23390 from postnatal day 4 to 20 also did not alter [3H]SCH-23390 binding or levels of D1 receptor mRNA. However, adult rats treated chronically with SCH-23390 exhibited increased [3H]SCH-23390 binding but did not show a significant change in D1 receptor mRNA levels.