Elena Battenberg

Transgenic mice expressing β-galactosidase in mature neurons under neuron-specific enolase promoter control

To gain insights into transcription factors defining neuronal identity, we generated transgenic mice carrying a 1.8 kb rat neuron-specific enolase (NSE) promoter fragment fused to an E. coli lacZ gene. Four of seven transgenic families expressed transgene RNA in the nervous system but not in most other tissues. Histochemical analysis of adult brain from the two lines with highest lacZ mRNA levels showed neuron-specific, pan-neuronal beta-galactosidase activity. Developmental RNA and histochemical analyses showed parallel onset of transgene and endogenous NSE gene expression in various neuronal cell types, although the magnitude of NSE mRNA accumulation later in development was not matched by the transgene. These results suggest that cis-acting regulatory elements, subject to neuron-specific control, are located within 1.8 kb upstream from the NSE gene.

Corticotropin releasing factor (CRF): immunoreactive neurones and fibers in rat hypothalamus

Abstract Antibodies to synthetic ovine corticotropin releasing factor (CRF) were used to localize nerve fibers and cell bodies in rat brain. Cell bodies, located nera the paraventricular nucleus, send a rich fiber plexus to the median eminence and less dense fiber connections to other hypothalamic and midbrain targets.

Endorphins are located in the intermediate and anterior lobes of the pituitary gland, not in the neurohypophysis.

Abstract Immunocytofluorescence techniques with well characterized anti-sera to α-endorphin and β-endorphin show presence of these two peptides in all cellular elements of the pars intermedia of the rat hypophysis, and in discrete cells of the pars distalis (adenohypophysis) at the complete exclusion of the neurohypophysis (pars nervosa, posterior lobe).

Hypothalamic enkephalin neurones may regulate the neurohypophysis.

WE have previously reported that significant amounts of immunoreactive (ir)-Leu5-enkephalin were present in extracts of the neurointermediate lobe of the rat pituitary1. Negligible amounts of the pentapeptide were detected in the anterior lobe. In these assays, the concentration of Leu5-enkephalin in the neurointermediate lobe was higher than in the globus pallidus, the brain region reported to contain the densest enkephalinergic innervation2. The high content of (ir)-Leu-enkephalin in the neurointermediate lobe of the pituitary led us to further investigation of its distribution and possible function. We report here that (ir)-enkephalins in the pituitary are concentrated in nerve fibres projecting from the hypothalamus to the pars nervosa and that this pathway may be involved in the regulation of neurohypophysial neurosecretion.

Noradrenergic Stimulation of Cyclic Adenosine Monophosphate in Rat Purkinje Neurons: An Immunocytochemical Study

A specific immunofluorescent histochemical method for cyclic adenosine monophosphate was used to study rat cerebellum. After topical treatment with norepinephrine or stimulation of norepinephrine-containing afferents from locus coeruleus, there was a striking increase in the number of Purkinje cells with strong cyclic adenosine monophosphate reactivity. Other putative inhibitory transmitters had no significant effect on staining of Purkinje cells. The results provide the first histochemical support for the hypothesis that cyclic adenosine monophosphate can be generated postsynaptically in central neurons in response to noradrenergic stimuli.

Distribution of neurons expressing immunoreactivity for the 5HT3 receptor subtype in the rat brain and spinal cord

The cellular distribution of the type 3 serotonin receptor (5HT3R) in the rat brain was established immunocytochemically by using a polyclonal antibody raised against a synthetic peptide from the deduced amino‐acid sequence of the cloned 5HT3R. The 5HT3R‐immunoreactive neurons were found in the forebrain, brainstem, and spinal cord, but within each region, the intensity of the immunoreactivity differed considerably. Within the forebrain, intensely immunoreactive cells were found in layers II–III of the neocortex, anterior olfactory nucleus, hippocampal formation, and amygdala. A few strongly immunoreactive neurons were consistently observed in the caudate putamen, and moderately or weakly labeled neurons were occasionally found in the nucleus accumbens. Within the brainstem, intensely labeled neurons were found in the trigeminal motor (V) and facial (VII) nuclei. Immunostained neurons were detected in the dorsal and the ventral horn of the spinal cord. These results reveal that the 5HT3R‐immunoreactive neurons are broadly distributed throughout the rat brain spinal cord, and suggest that this receptor can subserve significant participation in central nervous system neurotransmission. J. Comp. Neurol. 402:385–401, 1998.

The type 3 serotonin receptor is expressed in a subpopulation of GABAergic neurons in the rat neocortex and hippocampus

We used in situ hybridization and immunocytochemistry to investigate the presence of GABA in neurons that express the type 3 serotonin receptor (5-HT3R). Quantitative analysis indicated that more than 90% of 5-HT3R expressing cells are GABAergic in the neocortex and hippocampus. The co-existence of 5-HT3R and GABA in cortical and hippocampal neurons indicates that serotonin, via 5-HT3R, can affect GABA release and suggests the participation of 5-HT3R in the inhibitory regulation of forebrain neurons.

β-Endorphin: cellular localization, electrophysiological and behavioral effects

Tremendous excitement has been generated by the isolation, purification, and subsequent synthesis of the opioid peptides. Our collaborative efforts have been directed at the questions of where β-endorphin is stored in brain and pituitary, how such structures are related to those storing the enkephalins and possibly other biogenic substances, and the functions of these peptides expressed through their electrophysiological properties. Although none of these questions is yet completely answered, sufficient data have been accumulated (Bloom et al., 1976, 1977a, b; Guillemin et al., 1977a, b, c; Rossier et al., 1977a, b, c; Nicoll et al., 1977; Henriksen et al., 1977; French et al., 1977) to enable us to give this overview and progress report.

Cellular and subcellular immunolocalization of the type 3 serotonin receptor in the rat central nervous system

We developed and characterized 14 polyclonal antibodies against peptides whose sequences were predicted from the type 3 serotonin receptor subunit A (5-HT3R-A) cDNA. One such antiserum, 0165, raised against a peptide corresponding to the large putative intracellular loop, immunoprecipitated in vitro translated 5-HT3R-A protein and recognized both recombinant and neuronal 5-HT3R-A protein by Western blot at a high titer. Furthermore, when antiserum 0165 was used to immunolabel brain sections previously hybridized with a riboprobe specific for 5-HT3R-A transcripts, neuronal co-localization of immunoproduct and transcript was widely found throughout the brain. The study of the distribution of 5-HT3R-A-immunoreactivity in the rat central nervous system with antiserum 0165 revealed intensely immunolabeled neurons in the forebrain (isocortex, olfactory regions, hippocampal formation and amygdala), brainstem (sensory and motor nuclei and nuclei of the reticular formation) and spinal cord (dorsal and ventral horn). At the subcellular level, the 5-HT3R-A was found in endomembranes involved in translation (nuclear envelope and endoplasmic reticulum) and in the dendritic plasma-membrane. The present report is the first description of the 5-HT3R-A immunolocalization in the CNS. The wide distribution of the 5-HT3R-A in the brain and spinal cord based on ligand binding, in situ hybridization and immunolocalization studies support its participation in a large array of central nervous system functions.

Learning impairment in transgenic mice with central overexpression of corticotropin-releasing factor

The present studies were designed to test the learning and memory capacities of transgenic mice with central overexpression of corticotropin-releasing factor in a forced alternation water T-maze task and in the Morris water maze. In T-maze testing, littermate control mice reached a criterion of 70% correct responses after five days of trials, while the performance of transgenic subjects was still random after the same training. In Morris maze testing, control subjects reached the submerged platform significantly faster (F(1.48) = 4.51, P < 0.05) after three days of trials, while the performance of transgenic mice was unimproved over the same period. The deficit in Morris maze performance in transgenic mice was reversed when the platform was visible above the surface of the water. Pre-test administration of the benzodiazepine anxiolytic, chlordiazepoxide (10 mg/kg), before acquisition training also produced a significant (F(4.40) = 16.61, P < 0.001) and persistent improvement in Morris maze performance in transgenic mice when compared to vehicle-treated transgenic litter mates. Finally, there was no evidence of hippocampal cell loss in transgenic brains. The results suggest that corticotropin-releasing factor-overexpressing mice exhibit a profound learning deficit without sensory or motor-related impairments, and that memory plasticity can be restored by anxiolytic pre-treatment. Thus, constitutive overabundance of brain corticotropin-releasing factor may produce hyperemotionality that interferes with learned behaviors. Stress-related disorders characterized by co-morbid deficits in learning/memory may benefit from pharmacological normalization of brain corticotropin-releasing factor systems.