Piers C. Emson

Heterodimerization is required for the formation of a functional GABAB receptor

GABA (γ-aminobutyric acid) is the main inhibitory neurotransmitter in the mammalian central nervous system, where it exerts its effects through ionotropic (GABAA/C) receptors to produce fast synaptic inhibition and metabotropic (GABAB) receptors to produce slow, prolonged inhibitory signals. The gene encoding a GABAB receptor (GABABR1) has been cloned; however, when expressed in mammalian cells this receptor is retained as an immature glycoprotein on intracellular membranes and exhibits low affinity for agonists compared with the endogenous receptor on brain membranes. Here we report the cloning of a complementary DNA encoding a new subtype of the GABAB receptor (GABABR2), which we identified by mining expressed-sequence-tag databases. Yeast two-hybrid screening showed that this new GABABR2-receptor subtype forms heterodimers with GABABR1 through an interaction at their intracellular carboxy-terminal tails. Upon expression with GABABR2 in HEK293T cells, GABABR1 is terminally glycosylated and expressed at the cell surface. Co-expression of the two receptors produces a fully functional GABAB receptor at the cell surface; this receptor binds GABA with a high affinity equivalent to that of the endogenous brain receptor. These results indicate that, in vivo, functional brain GABAB receptors may be heterodimers composed of GABABR1 and GABABR2.

Striatal interneurones: chemical, physiological and morphological characterization

The neostriatum is the largest component of the basal ganglia, and the main recipient of afferents to the basal ganglia from the cerebral cortex and thalamus. Studies of the cellular organization of the neostriatum have focused upon the spiny projection neurones, which represent the vast majority of neurones, but the identity and functions of interneurones in this structure have remained enigmatic despite decades of study. Recently, the discovery of cytochemical markers that are specific for each of the major classes of striatal interneurones, and the combination of this with intracellular recording and staining, has revealed the identities of interneurones and some of their functional characteristics in a way that could not have been imagined by the classical morphologists. These methods also suggest some possible modes of action of interneurones in the neostriatal circuitry.

Distribution of vasoactive intestinal polypeptide in the rat and mouse brain.

Abstract The distribution of cell bodies and nerve fibers that combine with antisera to vasoactive intestinal polypeptide (VIP) was studied by immunohistochemistry in combination with radioimmunoassay in the brain of rat and mouse. The highest concentrations (60pmol/g wet wt) of immuno-reactive VIP were found in the cerebral cortex and in certain limbic structures, whereas the concentrations in the basal ganglia, thalamus, lower brain stem, cerebellum and spinal cord were low ( VIP-immunoreactive fibres had a distribution which on the whole paralleled that of the cell bodies, suggesting that many of the VIP-containing cells project locally. VIP-containing fibres were numerous in the following areas: the entire neocortex, the pyrifom cortex, the entorhinal cortex, the hippocampal complex, the amygdala (the central nucleus in particular), the anterior olfactory nuclei, the nucleus accumbens, ventral pallidum, bed nucleus of stria terminalis, suprachiasmatic nucleus, medial preoptic nucleus, median eminence, lateral geniculate body, pretectum, superior colliculus, periaqueductal gray, and the lateral parabrachial nucleus. Only few, scattered fibres were seen in other parts of the brain stem, in the striatum, thalamus and spinal cord. The cerebellum was devoid of VIP-containing fibres. VIP-containing neurones seem to form predominantly local projections. In addition, some VIP-containing neurones probably also form long projections, such as descending and transcallosal projections from the cortical cells, and projections from the amygdala to preoptic, hypothalamic and basal forebrain areas. The characteristic telencephalic distribution of the neurones that contain VIP suggests a role for this peptide in cortical and limbic functions.

Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin.

The temporal course of changes in peptide expression in the dorsal root ganglia L4 and L5 and in the dorsal horn of the spinal cord has been studied in rats subjected to a sciatic nerve transection at a mid-thigh level following different survival times. Galanin-, substance P-, vasoactive intestinal polypeptide-, peptide histidine-isoleucine- and calcitonin gene-related peptide-like immunoreactivities have been studied both by immunohistochemistry and radioimmunoassay. Galanin messenger ribonucleic acid has also been studied by in situ hybridization in the dorsal root ganglia of normal and lesioned animals. In addition, a group of animals with a sciatic nerve crush was studied to compare possible differences in peptide expression after both types of lesions. The results show that the transection induces an increase in the number of cell bodies expressing galanin-like immunoreactivity in the ganglia, and that the galanin levels rise about 120-fold after three and 14 days of survival. This increase reflected increased synthesis of the peptide, since there was a rise in the galanin messenger ribonucleic acid already at 24 h post-lesion, which was maintained for at least 60 days. In the spinal cord there was an increase of staining in the midportion of the outer layers of the dorsal horn that corresponded to fibers thought to arise from cells of the dorsal root ganglia affected by the transection. Also a depletion of substance P-like and an increase in vasoactive intestinal polypeptide- and peptide histidine-isoleucine-like immunoreactivities in the dorsal root ganglia were confirmed. These changes were shown to be rapidly detectable and were paralleled by similar changes in the dorsal horn of the spinal cord. For calcitonin gene-related peptide the immunohistochemistry was inconclusive, and the radioimmunoassay showed no detectable changes. After nerve crush a transient increase in the number of galanin immunoreactive neurons was observed, as well as a decrease in the number of neurons showing substance P-like immunoreactivity. These changes were most noticeable between six and 14 days of survival. After this, peptide expression seemed to return slowly to normal, that is by day 45 post-crush only a few cells showed galanin-like, and many sensory neurons expressed substance P-like immunoreactivity. The results demonstrate that when primary sensory neurons are peripherally lesioned they respond in a complex manner, altering their normal production of peptides by increasing or decreasing their synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Two classes of cortical GABA neurons defined by differential calcium binding protein immunoreactivities

SummaryCalcium ions play a key role in many aspects of neuronal behavior and certain calcium binding proteins that may influence this behavior are differentially distributed in the central nervous system. In this study it is shown that immunoreactivity for calbindin-28 and for parvalbumin is localized in separate populations of inhibitory GABA interneurons in all areas of the neocortex of Old World monkeys. Virtually all GABA neurosn show immunoreactivity for one or other calcium binding protein but, except for a few cells in layer IV, GABA cells do not show immunoreactivity for both proteins. Among the two cell populations, parvalbumin immunoreactivity characterizes basket neurons while calbindin immunoreactivity characterizes double bouquet neurons. These findings suggest that the two GABA cell types differ in their regulation of calcium homeostasis and may yield clues to their different roles in intracortical circuitry.

Abundant Tau Filaments and Nonapoptotic Neurodegeneration in Transgenic Mice Expressing Human P301S Tau Protein

The identification of mutations in the Tau gene in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has made it possible to express human tau protein with pathogenic mutations in transgenic animals. Here we report on the production and characterization of a line of mice transgenic for the 383 aa isoform of human tau with the P301S mutation. At 5–6 months of age, homozygous animals from this line developed a neurological phenotype dominated by a severe paraparesis. According to light microscopy, many nerve cells in brain and spinal cord were strongly immunoreactive for hyperphosphorylated tau. According to electron microscopy, abundant filaments made of hyperphosphorylated tau protein were present. The majority of filaments resembled the half-twisted ribbons described previously in cases of FTDP-17, with a minority of filaments resembling the paired helical filaments of Alzheimers disease. Sarkosyl-insoluble tau from brains and spinal cords of transgenic mice ran as a hyperphosphorylated 64 kDa band, the same apparent molecular mass as that of the 383 aa tau isoform in the human tauopathies. Perchloric acid-soluble tau was also phosphorylated at many sites, with the notable exception of serine 214. In the spinal cord, neurodegeneration was present, as indicated by a 49% reduction in the number of motor neurons. No evidence for apoptosis was obtained, despite the extensive colocalization of hyperphosphorylated tau protein with activated MAP kinase family members. The latter may be involved in the hyperphosphorylation of tau.

Neuropeptide Y: Cerebrovascular innervation an vasomotor effects in the cat ☆

Avian pancreatic polypeptide (APP) has been proposed to be a neurotransmitter in a subpopulation of sympathetic nerves. Here, we present immunocytochemical and pharmacological evidence that the structurally related peptide, neuropeptide Y (NPY), is likely to be the biologically active material in these nerves. Cerebral arteries from cats are invested with a dense network of NPY-containing nerve fibres, as demonstrated by immunocytochemistry. This immunoreaction is abolished by prior removal of the superior cervical ganglion. NPY causes strong contractions of cerebral arteries in vitro whereas APP has small effects on the vasomotor reactivity. The NPY-induced contractions were not inhibited by the alpha 2-adrenoceptor antagonist rauwolscine (10(-7) M) or the 5-hydroxytryptamine antagonist ketanserin (10(-7) M). The contractions were, however, sensitive to calcium removal or to the calcium antagonist diltiazem (10(-4) M).

Relationship of neuronal nitric oxide synthase immunoreactivity to GnRH neurons in the ovariectomized and intact female rat.

The present study has used a rat neuronal nitric oxide synthase (nNOS) antibody to examine the relationship of nNOS immunoreactivity to GnRH neurons in the ovariectomized and intact diestrous and proestrous rat. A striking band of nNOS‐immunoreactive cells was identified in the rostral preoptic area which began in the median preoptic nucleus and organum vasculosum of the lamina terminalis and formed an inverted Y‐type distribution above the rostral third ventricle at the level of the anteroventral periventricular nucleus. Another band of nNOS‐immunoreactivity was found extending through the internal zone of the median eminence into the arcuate nucleus. Although nNOS immunoreactivity was not detected within GnRH neuronal cell bodies in any of the experimental groups, GnRH perikarya located in the rostral preoptic area, but not elsewhere, were found to be surrounded by nNOS‐containing cells. In the median eminence, nNOS and GnRH immunoreactivities were distributed separately in the internal and external zones, respectively.

Topographic projections of substance P and GABA pathways in the striato- and pallido-nigral system: A biochemical and immunohistochemical study

The topographical projections of substance P pathways from the caudateputamen and globus pallidus to the pars compacta and pars reticulata of the substantia nigra have been investigated in the rat using immunohistochemical and radioimmunoassay techniques and compared with the projections of GABA nergic striatal neurones. Unilateral vertical knife cuts through the anterior and posterior striatum have shown the majority of substance P-containing neurones which project to the substantia nigra to originate in the most rostral part of the caudate-putamen. This projection appears to innervate the pars reticulata and pars compacta of the substantia nigra to a similar extent. A separate projection of substance P-containing neurones to the substantia nigra appears to originate in the globus pallidus. Undercutting the cerebral cortex which overlies the corpus striatum did not affect the substance P content of the globus pallidus or substantia nigra. However, there appears to be an additional substance P projection from the basal ganglia to the entopeduncular nucleus. In contrast, GABA-containing neurones which project to the substantia nigra are mainly located in more caudal parts of the caudate-putamen and in the globus pallidus. There is a marked differentiation in the region of the substantia nigra innervated by GABA cells originating in the rostral and caudal parts of the corpus striatum. Rostrally situated neurones project almost exclusively to the pars reticulata, while neurones in the caudal part of the caudate-putamen and globus pallidus project to both the pars compacta and pars reticulata. These results suggest that there is a partial topographical separation of the sites of origin of substance P- and GABA-containing neurones which project to the substantia nigra.

AChE-stained horizontal sections of the rat brain in stereotaxic coordinates.

This paper presents an atlas of the rat brain based on horizontal sections stained for acetylcholinesterase (AChE). Fourteen labeled photographs from sections at 0.5 mm intervals are presented. The atlas is intended for use with male rats 250-300 g in weight. It was constructed using the flat-skull position (lambda and bregma in same horizontal plane). Either the interaural midline or bregma can serve as the reference zero point. We have found the stain for AChE to be a useful general purpose stain for identifying brain nuclei and tracts.