Roland D. Ciaranello

Immunocytochemical localization and description of neurons expressing serotonin2 receptors in the rat brain

Serotonin2 receptors have been implicated in a variety of behavioral and physiological processes, as well as a number of neuropsychiatric disorders. To specify the brain regions and specific cell types possessing serotonin2 receptors, we conducted an immunocytochemical study of the rat brain using a polyclonal serotonin2 receptor antibody. Perfusion-fixed rat brain sections were processed for immunocytochemistry and reactivity was visualized using an immunoperoxidase reaction. Numerous small, round neurons were heavily labeled in the granular and periglomerular regions of the olfactory bulb. Heavy labeling of medium-sized multipolar and bipolar neurons was also seen in olfactory regions of the ventral forebrain, including the anterior olfactory nucleus and olfactory tubercle. Other regions of the basal forebrain exhibiting high levels of immunoreactivity were the nucleus accumbens, ventral pallidum, Islands of Calleja, fundus striatum and endopyriform nucleus. Immunoreactive neurons were also seen in the lateral amygdala. A dense band of small, round cells was stained in layer 2 of pyriform cortex. In neocortex, a very sparse and even distribution of bipolar and multipolar neurons was seen throughout layers II-VI. A much more faintly labeled population of oval cells was observed in the deep layer of retrosplenial and posterior cingulate cortex, and in the granular layer of somatosensory frontoparietal cortex. A moderate number of medium bipolar and multipolar cells were scattered throughout the neostriatum, and a moderate number of pyramidal and pyramidal-like cells were seen in the CA fields of the hippocampus. Diencephalic areas showing immunolabeling included the medial habenula and anterior pretectal nucleus, with less labeling in the ventral lateral geniculate. In the hindbrain, two dense populations of large multipolar cells were heavily labeled in the pedunculopontine and laterodorsal tegmental nuclei, with lesser labeling in the periaqueductal gray, superior colliculus, spinal trigeminal nucleus and nucleus of the solitary tract. Based on the distribution, localization and morphology of immunoreactive neurons in these regions, we hypothesize that subpopulations of serotonin2 containing cells may be GABAergic interneurons or cholinergic neurons. Further, the observed distribution suggests that the physiological effects of serotonin acting through serotonin2 receptors are mediated by a relatively small number of cells in the brain. These observations may have strong functional implications for the pharmacological treatment of certain neuropsychiatric disorders.

Isolation and characterization of a novel cDNA clone encoding a homeodomain that is developmentally regulated in the ventral forebrain

A complementary DNA, Tes-1, of a novel homeodomain protein has been cloned, and its pattern of expression has been characterized. It is a structural homolog of Distal-less, a homeodomain-encoding gene in D. melanogaster. Its expression is developmentally regulated and is limited to structures in the head. Within the central nervous system of the midgestation mouse embryo, it is expressed exclusively in the ventral forebrain. It is likely that Tes-1 plays a regulatory role in the development of this complex neural structure.

Developmental regulation of 5-HT2 and 5-HT1c mRNA and receptor levels.

We investigated the regulation of 5-HT2 and 5-HT1c receptors and their corresponding mRNAs during rat brain development. This study showed that 5-HT2 and 5-HT1c receptors increased markedly during ontogeny. 5-HT2 receptors, measured with [3H]ketanserin or [125I]lysergic acid diethylamide binding, increased 8-fold between embryonic day 17 (E17) and postnatal day 13 (P13). 5-HT2 receptor mRNA levels, quantified by probing Northern blots of total RNA with a synthetic oligonucleotide cDNA probe, multiplied 13-fold between E17 and P5. The developmental pattern of 5-HT2 receptor and mRNA expression appeared to correlate with the serotonergic hyperinnervation of the cortex which occurs between P2 and P17. 5-HT1c receptors, measured with [125I]lysergic acid diethylamide under site-specific conditions, increased 2-fold between E17 and P27, 5-HT1c mRNA increased 5-fold between E17 and P27. Interestingly, the developmentally induced variations in 5-HT1c receptors did not precisely correlate with mRNA alterations. Further study of the factors responsible for these alterations could help to explain the molecular and biochemical mechanisms responsible for modulating receptor levels in vivo.

The Neurobiology and Genetics of Infantile Autism

Autism is a syndrome with multiple etiologies, as is made clear both by the evidence of neurobiological research and by the catalog of disorders that present with autistic behaviors. What remains unclear are the specific neuropathological mechanisms that produce autistic behaviors; for example, is there a common neuroanatomic pathology for all cases of autism, or can autistic behaviors emerge from different pathological sequences within the brain? Although it is premature to generalize, neuropathological studies appear to have identified common abnormalities in the cerebellum and limbic system of at least five autistic subjects. These subjects, with variable levels of mental retardation, demonstrated marked Purkinje cell loss in the cerebellar hemispheres, together with retained fetal neuronal circuitry in cerebellar nuclei and increased neuronal packing in specific regions of the limbic system, amygdala, and hippocampus. The architecture of the cerebral cortex was not affected. Although our knowledge of brain functioning is incomplete, alterations of the kind noted in the cerebellum and limbic system could reasonably produce autistic behaviors. For more detail, readers are directed to a review of cerebellar contributions to higher functions by Schmahmann (1991). Neuroimaging studies allow less resolution of brain structure than do neuroanatomic studies, and the reported findings from neuroimaging are somewhat contradictory. However, a number of investigators have reported structural abnormalities in ventricle size and cerebral hemispheric asymmetry using CT. MRI, which offers greater resolution, has uncovered some consistent findings, along with a variety of nonspecific abnormalities. Common abnormalities include reduced volume of cerebellar hemispheres and vermal lobules--findings not inconsistent with the above-mentioned neuropathological defects. It is also interesting to note that individuals with fragile X syndrome have similar cerebellar findings. PET and NMR studies of autism are at a preliminary stage, but these methodologies allow insight into the functioning of the brain, rather than simply brain anatomy. Recent PET studies indicating decreased association between paired regions of the brains of autistic subjects are of interest, particularly if they can be confirmed and refined by additional studies. Neurophysiological studies also offer insight into brain function, but are subject to numerous methodological criticisms. Nevertheless, recent reports of diminished P300 waves and absent NC components in autistic subjects seem to indicate fundamental defects in attention and secondary processing, which could help explain the self-stimulatory behaviors often seen in autism. The disturbances in brain development associated with autism can be produced in a number of ways, and at different times during development of the nervous system.(ABSTRACT TRUNCATED AT 400 WORDS)

Co-Localization of substance P- and phenylethanolamine-N-methyltransferase-like immunoreactivity in neurons of ventrolateral medulla that project to the spinal cord: Potential role in control of vasomotor tone

Both substance P (SP)- and epinephrine-containing neurons in the rostral ventrolateral medulla have been thought to play a role in regulating vasomotor tone. The combination of retrograde transport of a fluorescent dye (Fast Blue) and immunofluorescent staining for SP- and phenylethanolamine-N-methyltransferase (PNMT)-immunoreactivity was used to determine the relationships of these two groups of ventrolateral medullary neurons which project to the spinal cord. The majority of spinally projecting neurons in the rostral ventrolateral medulla contain both PNMT-like and SP-like immunoreactivity. The presence of PNMT-immunoreactive material in a neuron implies that epinephrine is a probable neurotransmitter for such a cell. Earlier work demonstrated that epinephrine and SP have opposite effects on the firing of sympathetic preganglionic neurons. Our results raise the possibility of a novel mechanism of synaptic regulation of the sympathetic preganglionic vasomotor neurons.

Catecholamines, strain differences in biosynthetic enzyme activity in mice.

Abstract In five strains of inbred mice, activities of phenylethanolamine N -methyl transferase and tyrosine hydroxylase and amounts of norepinephrine and epinephrine in the adrenal gland vary markedly. Tyrosine hydroxylase activity in the brain also varies widely. The fact that these measures vary independently of each other suggests that several genetic factors are involved. The finding of genetic differences affecting catecholamine synthesis has implications for the study of catecholamine regulatory mechanisms and the relationship between catecholamines and behavior.

Empirically derived subclassification of the autistic syndrome

A method is presented for empirical subclassification of autistic and autisticlike children, based on observations of current behavior. The advantage of the method is that it identifies profiles of cooccurring behaviors and accordingly assigns children to subtypes. The subtypes are more clinically homogeneous than the overall sample of autistic children. Preliminary findings are presented, including an effort to validate the subclasses by suggesting possible relationships between subtype membership and perinatal markers, developmental milestones, and independent measures of concurrent behavior.

Narcolepsy: Cholinergic receptor changes in an animal model

An inbred colony of narcoleptic doberman pinschers has been analyzed for muscarinic receptor levels in 19 discrete brain regions. In comparison to age-matched controls, receptors were generally elevated in the brainstem and reduced in forebrain areas. No changes in receptor binding affinity were detected. The increased receptor levels found in the brainstem suggest that cholinoceptive neurons in this region are hypersensitive and may be involved in the initiation of cataplexy and other aspects of the narcolepsy syndrome.

Transcriptional regulation of hippocampal 5-HT1a receptors by corticosteroid hormones

5-HT1a receptors in the hippocampus play a critical role in modulating limbic system output. The activity and level of 5-HT1a receptors are modulated by glucocorticoid levels. The present study was undertaken to test the hypothesis that glucocorticoids attenuate the transcriptional activity of the 5-HT1a receptor gene. Using in situ hybridization and RNase protection assays, we observed a substantial increase in 5-HT1a mRNA expression after adrenalectomy in the same hippocampal regions in which 5-HT1a binding sites are increased. This increase in 5-HT1a mRNA expression occurs as early as 1 h after adrenalectomy and precedes the increase in receptor binding sites. Further in situ hybridization analysis showed that 5-HT1a mRNA is increased within individual hippocampal cells after adrenalectomy. Administration of dexamethasone completely prevents the adrenalectomy-induced elevation in hippocampal 5-HT1a receptor mRNA. Nuclear run-on assays showed that the rate of transcription of 5-HT1a mRNA after adrenalectomy increased 70% above the rate from control preparations and could be reduced to basal levels by the administration of dexamethasone. Adrenalectomy did not cause an increase in functional coupling of 5-HT1a receptors to adenylyl cyclase or phospholipase C. These results suggest that transcription of hippocampal 5-HT1a receptor mRNA is under negative regulation by corticosteroid hormones.

Neurons Expressing 5-HT2 Receptors in the Rat Brain: Neurochemical Identification of Cell Types by Immunocytochemistry

The serotonin2(5-HT2) receptor has been implicated in a number of behavioral and physiological processes. It may also play a role in cellular development and differentiation, and represents a site of action of hallucinogens and certain psychotherapeutic drugs. To better understand the functions and regulation of the 5-HT2 receptor, we have undertaken a series of studies in which we attempted to identify the specific cell types that express the receptor. This was accomplished using a variety of double-labeling strategies with an antibody we raised against the rat 5-HT2 receptor protein. In this review, we recount of some of our previously published findings and present some new data in which we identify subpopulations of cholinergic neurons in the brainstem and gamma-aminobutynic acid (GABA)ergic interneurons in the cortex that express 5-HT2 receptor immunoreactivity. Developmentally, the appearance of 5-HT2 receptor immunoreactivity occurs relatively late in the ontogeny of the cells in which it is expressed, mostly in the early postnatal period. This argues against a significant role for this receptor in early development, though it may participate in some aspect of terminal differentiation. We discuss the significance of the cell-type-specific and temporal expression of the 5-HT2 receptor in the context of current hypotheses of neuropsychiatric disorders such as schizophrenia.