bluuebunny/crossref_metadata_2025 huggingface dataset:
Compartmentation and glycoprotein substrates of calpain in the developing rat brain
Authors: Sheppard Allan , Wu Justina , Bahr Ben A. , Lynch Gary
Published: December 2004

An activated form of calpain I associates with telencephalic membranes in a developmentally regulated fashion during early postnatal ontogeny. During this period, the cytoskeletal component spectrin is available and appears to be differentially susceptible to calpain‐mediated cleavage. Lectin blotting techniques demonstrated that the leupeptin‐sensitive action of calpain is primarily directed toward large proteins which are glycoconjugate in nature; neuronal cell adhesion molecules are among the glycoproteins whose associations with the telencephalic membranes decrease due to calpain activity. These data suggest that cytoplasmic calpain is translocated to the membrane during early brain development in order to act on the cytoskeletal and adhesive structures responsible in part for neuronal shape and function.
200 kD neurofilament protein and synapse elimination in the rat soleus muscle
Authors: Roden R. L. , Donahue S. P. , Schwartz G. A. , Wood J. G. , English A. W.
Published: December 2004

We studied the distribution and appearance of the phosphorylated form of the 200 kD neurofilament protein in the rat soleus muscle during the period of postnatal synapse elimination. Unlike many muscles, the appearance of singly innervated muscle cells in soleus occurs well after myogenesis has been completed, so that synapses are eliminated from a stable population of muscle cells. Immunoreactivity to the 200 kD neurofilament protein is present in the terminals of neuromuscular synapses of animals at all postnatal ages from 0 to 21 days. Before postnatal day 10, when physiological studies indicate that all soleus muscle cells receive more than one synaptic input, as many as 30% of soleus muscle cells contain phosphorylated 200 kD neurofilament protein immunoreactivity in only one synaptic terminal. At older ages the number of polyneuronally innervated muscle cells observed using immunostaining is similar to that observed physiologically. These findings suggest that not all developing neuromuscular synapses contain phosphorylated 200 kD neurofilament protein, and that those terminals lacking it comprise most of those eliminated early in the postnatal period. We conclude that the presence of phosphorylated 200 kD neurofilament protein might be highly correlated with the survival of motor nerve terminals during postnatal neuromuscular synapse elimination.
Arylamine spider toxins antagonize NMDA receptor‐mediated synaptic transmission in rat hippocampal slices
Authors: Mueller Alan L. , Albensi Benedict C. , Ganong Alan H. , Reynolds Linda S. , Jackson Hunter
Published: December 2004

The effects of arylamine spider toxins on synaptic transmission in rat hippocampal slices were investigated. Two different responses were monitored: the AMPA receptor‐mediated population spike recorded in control buffer (selectively antagonized by DNQX) and the NMDA receptor‐mediated EPSP recorded in nominally magnesium‐free buffer containing 20 μM DNQX (selectively antagonized by AP5, AP7, and dizocilpine (MK‐801)). The synthetic arylamine spider toxins JSTX‐3, argiotoxin‐636, and argiotoxin‐659 were 26 to 73 times more potent at antagonizing the NMDA receptor‐mediated EPSP (IC50 values ranging from 12 to 24 μM) than the AMPA receptor‐mediated population spike (IC50 values ranging from 612 to 878 μM). These results indicate that arylamine spider toxins are selective antagonists of NMDA receptors in the mammalian CNS.
Action of serotonin in the medial prefrontal cortex: Mediation by serotonin3‐like receptors
Authors: Ashby Charles R. , Edwards Emmeline , Wang Rex Y.
Published: December 2004

In the present study, we investigated the effects of various serotonin (5‐HT) antagonists on 5‐HT's action on medial prefrontal cortical cells (mPFc) using the techniques of single cell recording and microintophoretic The microiontophoretic application of 5‐HT (10–80 nA) produced a current‐dependent suppression of mPFc cell firing and this effect was blocked by the selective 5‐HT3 receptor antagonists (±)‐zacopride, ICS 205930 and granisetron at currents of 5–20 nA. Furthermore, the intravenous (i.v.) administration of (±)‐zacopride (5–50 μg/kg) markedly attenuates the suppressive action of 5‐HT on mPFc cell firing. In contrast, the microinotophoresis of 5‐HT1 and 5‐HT2 receptor antgonists such as (±)‐pindolol, spiperone, metergoline, and ritanserin (10–20 nA) failed to block 5‐HT's effect. In fact, in some cells, spiperone and ritanserin potentiated 5‐HT's action and prolonged neuronal recovery. In addition, the intravenous administration of either ritanserin (5–2,000 μg/kg) or metergoline (4–2,400 μg/kg) failed to alter 5‐HT's action. The electrical stimulation of the caudal linear raphe nucleus (CLi) suppressed the spontaneous activity of 83% of the mPFc cells tested by 45 ± 2%. This suppression was significantly attenuated by the iontophoresis of granisetron (2.5–5 nA) but not by the 5‐HT2 and 5‐HT1C receptor antagonist ritanserin or the relatively selective 5‐HT2 receptor antagonist (+)‐MDL 11,939 (10–40 nA). However, the i.v. administration of ritanserin (0.5–1.5 mg/kg) or S‐zacopride (0.1 mg/kg) significantly blocked the suppression of mPFc cell firing produced by CLi stimulation. Overall, our results suggest that 5‐HT's physiological effect in the mPFc may be primarily mediated by its interaction with 5‐HT3‐like receptors and that ritanserin's blockade of CLi‐induced suppression of mPFc cell firing may be indirect.
Compartmental distribution of cytochrome oxidase in the striatum of the rat
Authors: Augood Sarah J. , Emson Piers C.
Published: December 2004

Endogenous cytochrome oxidase activity was investigated in the adult rat striatum at the light microscope level to see if it was distributed in accordance with the established striatal patch/matrix compartmentalisation. Striatal sections stained to visualise cytochrome oxidase activity were compared with serial sections stained to visualise tyrosine hydroxylase and calbindinD28k‐like immunoreactivity, established markers of the matrix compartment. The distribution of endogenous cytochrome oxidase activity was found to coincide with the immunocytochemical staining pattern seen for tyrosine hydroxylase and calbindinD28k whereby areas of intense tyrosine hydroxylase and calbindinD28k‐like immunoreactivity (termed the matrix) corresponded to areas of intense cytochrome oxidase activity. Conversely, areas of less intense tyrosine hydroxylase and calbindinD28k‐like immunoreactivity (termed patches) corresponded to areas of low cytochrome oxidase activity. In addition, the distribution of two other oxidative enzymes involved in the regulation of mitochondrial respiration, succinic dehydrogenase and NADH‐diaphorase, was examined in the striatum and substantia nigra by using histochemical techniques. Both NADH‐diaphorase and succinic dehydrogenase histochemistry showed an uneven pattern of neuropil staining in the striatum. In the substantia nigra a few intensely stained cell bodies were seen in the dorsal‐lateral tip of the pars reticulata with both histochemical techniques. By using an anti‐cytochrome oxidase antibody an abundance of immunoreactive cell bodies and processes were seen in the substantia nigra, particularly in the dorso‐medial rim and dorsal tip of the pars reticulata. The substantia nigra pars lateralis contained many intensely stained cytochrome oxidase‐like immunoreactive cell bodies and processes. Our results demonstrate that the neurochemically distinct compartments of the adult rat striatum contain differential amounts of endogenous cytochrome oxidase activity suggesting that the matrix compartment is more metabolically active than the patch compartment. This difference in metabolic activity may reflect a difference in synaptic activity between the two striatal compartments.
Cholinergic and GABAergic mediations of the effects of apomorphine on serotonin neurons
Authors: Chen H. Y. , Lin Y. P. , Lee E. H. Y.
Published: December 2004

Apomorphine (APO) has been shown to elevate tryptophan, serotonin (5‐HT), and 5‐hydroxyindoleacetic acid (5‐HIAA) concentrations in the dorsal raphe (DR) and its corresponding projection site, the striatum, but not in the median raphe (MR) and its terminal area, the hippocampus. We have previously demonstrated that these effects are indirectly mediated through dopamine (DA) autoreceptors in the substantia nigra and possibly γ‐aminobutyric acid (GABA) neurons in or near the DR. In the present study, we have further found that the effects of APO on 5‐HT neurons are also mediated through both nicotinic and M1 muscarinic cholinergic receptors as well as GABAA receptors in the DR. This suggestion is based on the findings that both atropine and mecamylamine antagonized the effects of APO, while carbachol at a high dose exerted an effect opposite to that of APO. Besides, pirenzepine and bicuculline at low doses also antagonized, whereas saclofen did not alter the influence of APO on 5‐HT in the striatum. Bicuculline at a higher dose enhanced tryptophan and 5‐HT measures by itself. None of the drugs studied had a significant effect on tryptophan, 5‐HT, or 5‐HIAA in the hippocampus. These results together suggest that DA, ACh, and GABA neurons are all involved in the actions of APO on 5‐HT, while the direct synaptic relationships among these neurotransmitters and the precise anatomical locus for these interactions to occur are still unknown. It is possible that APO, by inhibiting DA neuron firing in the substantia nigra and through the GABA disinhibition mechanism, therefore indirectly activates 5‐HT neurons in the DR and the striatum. While the above neuronal firing model well explains the elevation of 5‐HIAA, the simultaneous increases of tryptophan and 5‐HT, especially tryptophan, may be more readily explained by a mechanism of tryptophan uptake upon APO administration. Further anatomical, biochemical, and electrophysiological studies are ongoing to test this hypothesis and to clarify the circuit and the anatomical locus (loci) for these interactions to occur.
Multiple heteroreceptors on limbic thalamic axons: M2 acetylcholine, serotonn1B, β2‐adrenoceptors, μ‐opioid, and neurotensin
Authors: Vogt Brent A. , Crino Peter B. , Jensen Eugene L.
Published: December 2004

Ligand binding to many transmitter receptors is much higher in layer Ia of rat posterior cingulate cortex than it is in other layer, and this is where most axons from the anterior thalamus terminate. The present study explores the possibility that a number of receptors may be expressed on axons from limbic thalamic nuclei that terminate in layer Ia. Unilateral thalamic lesions were placed in rats and, 2 weeks later, five ligand binding protocols, coverslip autoradiography, and single grain counting techniques were used to qunatify binding in control and ablated hemispheres. Binding to the following receptor subtypes was analyzed: M2 acetylcholine, 3H‐oxotremorine‐M, or 3H‐AF‐DX 116 with 50 nM pirenzepine; serotonin1B, 125I‐(−)‐cyanopindolol with 30 μM isoproterenol; β2‐adrenoceptors, 125I‐(−)‐cyanopindolol with 1 μM serotonin and 10 μM atenolol; μ‐opioid, 3H‐Tyr‐D‐Ala‐Gly‐MePhe‐Gly‐ol; neurotensin, 3H‐neurotensin. Thalamic lesions reduced binding in two laminar patterns. In one pattern, there was a major reduction in binding in most superficial layers with that in layer Ia ranging from 50 to 70% for binding to M2 muscarinic and serotonin1B receptors. Binding to β2‐adrenoceptors was also reduced in most superficial layers but to a lesser extent. In the second pattern, reductions were limited to layer I with losses in layer Ia of 20–30% for μ‐opioid and neurotensin receptors. In no instance was layer Ia binding completely abolished (i.e., postlesion peaks remained).

Since the transmitters for each of the five receptors analyzed in this study are not synthesized by anterior or laterodorsal thalamic neurons, these receptors are heteroreceptors. The greatest postlesion reduction in M2 binding was for AF‐DX 116 and so most M2 heteroreceptors are of the “cardiac” subtype. Finally, the diverse population of heteroreceptors on limbic thalamic axons provides for presynaptic modulation by a wide range of transmitter systems and suggests that thalamocortical transmission may not be a simple, unmodulated event.
Local application of bicuculline potentiates NMDA‐receptor‐mediated sensory responses of brain noradrenergic neurons
Authors: Shiekhattar Ramin , Aston‐Jones Gary
Published: December 2004

Direct application of bicuculline methiodide (BIC) to noradrenergic locus coeruleus (LC) neurons potently enhanced their sensory responsiveness. This increased responsiveness was due to the long‐lasting expression of a new, N‐methyl‐D‐aspartate (NMDA) receptor‐mediated component of the synaptic response. This enhancement only occurred when a high stimulus intensity was used to induce the sensory response. A similar increase in responsiveness was observed with stimulation of the nucleus paragigantocellularis (PGi), one of the major direct afferents to LC. This action of BIC was neither mimicked by picrotoxin, penicillin, or the GABA‐B antagonist, 2‐hydroxysaclofen, nor by agents that directly depolarize LC neurons. In addition, the inverse agoinst of the benzodiazepine receptor, methyl‐6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate (DMCM), did not mimic this effect of BIC. The BIC‐potentiated response component was eliminated by direct application of the neurotransmitter γ‐aminobutyric acid (GABA). These results indicate that BIC, acting at a possibly novel site, unmasks NMDA receptors that can be activated by sensory stimuli. This may reflect a mechanism whereby interactions between two major neurotransmitter systems, excitatory amino acids (EAAs) and GABA, potently modulate signal transmission in the brain.
Activators of protein kinase C increase the phosphorylation of the synapsins at sites phosphorylated by cAMP‐dependent and Ca2+/calmodulin‐dependent protein kinases in the rat hippocampal slice
Authors: Browning Michael D. , Dudek Ellen M.
Published: December 2004

Previous studies have shown that activators of protein kinase C (C kinase) produce synaptic potentiation in the hippocampus. For example, the C kinase activator phorbol dibutyrate has been shown to increase transmitter release in the hippocampus. In addition, a role for C kinase in long‐term potentiation has been proposed. A common assumption in such studies has been that substrates for C kinase were responsible for producing these forms of synaptic potentiation. However, we have recently shown that phorbol dibutyrate increased the phosphorylation of synapsin II (formerly protein III, Browning et al., 1987) in chromaffin cells (Haycock et al., 1988). Synapsin II is a synaptic vesicle‐associated phosphoprotein that is a very poor substrate for C kinase but an excellent substrate for cAMP‐dependent and Ca2+/calmodulin‐dependent protein kinase. We felt, therefore, that activation of C kinase might lead to activation of a kinase cascade. Thus effects of C kinase activation might be produced via the phosphorylation of proteins that are not substrates for C kinase. In this report we test the hypothesis that activators of C kinase increase the phosphorylation of synapsin II and an homologous protein synapsin I. Our data indicate that PdBu produced dose‐dependent increases in the phosphorylation of synapsin I and synapsin II. We also performed phospho‐site analysis of synapsin I using limited proteolysis. These studies indicated that PdBu increased the phosphorylation of multiple sites on synapsin I. These sites have previously been shown to be phosphorylated by both cAMP‐dependent protein kinase and the multifunctional Ca2+/calmodulin‐dependent protein kinase II. The ability of C kinase activators to produce a net increase in the phosphorylation state of these sites on synapsin was confirmed by a “back phosphorylation assay” with exogenous cAMP‐ and Ca2+/calmodulin‐dependent protein kinases. Previous studies have provided direct evidence that synapsin I regulates neurotransmitter release at the squid giant synapse and that this effect is controlled by phosphorylation of synapsin I by Ca2+/calmodulin‐dependent protein kinase II. Accordingly we hypothesize that the phosphorylation of synapsin I and the related protein, synapsin II, may play an important role in mediating the increased transmitter release elicited by activators of C kinase. Moreover, we suggest that the role played by C kinase in long‐term potentiation may involve, in part, the phosphorylation of the synapsins.
Autoradiographic localization of 5HT1 binding sites in the medulla oblongata of the rat
Authors: Thor Karl B. , Blitz‐Siebert Alisa , Helke Cinda J.
Published: December 2004

Serotonin (5HT) binding sites in the medulla oblongata of the rat were localized using autoradiographic techniques with radioactive ligands that express high affinity for the 5HT1, (3H‐5HT), 5HT1A (3H‐8OH‐DPAT), or 5HT1B (125I‐CYP with isoproterenol) receptor subtypes. 5HT1A sites were concentrated primarily in certain sensory regions of the medulla and in regions that contain serotonergic neurons. 5HT1B sites were diffusely distributed throughout the reticular formation and motor regions as well as being localized to certain sensory regions. A surprising finding was an association of 5HT1B binding sites with the corticospinal tract. 3H‐5HT binding generally reflected the combined pattern of 5HT1A and 5HT1B sites but was also extremely dense in the choroid plexus, which exhibited virtually no 5HT1A or 5HT1B ligand binding. Presumably this binding, which was blocked by ketanserin, corresponds to 5HT1C sites.

Administration of the serotonergic neurotoxin 5,7‐dihydroxytryptamine reduced 5HT1A binding sites in regions of the medulla that contain serotonergic neuronal cell bodies. 5HT1B binding was not significantly altered in any area of the medulla.

These studies indicate an important role for 5HT1A ligands in the processing of visceral and somatic sensory information, in regulation of certain cerebellar afferent projections, and in the regulation of serotonergic neuronal activity. 5HT1B ligands would be expected to regulate visceral and somatic efferent activity, as well as sensory information and reticular efferent activity, and might presynaptically regulate cortical inputs to the brain stem and spinal cord.
Distribution of beta‐adrenergic receptor subtypes in human post‐mortem brain: Alterations in limbic regions of schizophrenics
Authors: Joyce Jeffrey N. , Lexow Nedra , Kim Soo Jin , Artymyshyn Roman , Senzon Shari , Lawerence David , Cassanova Manuel F. , Kleinman Joel E. , Bird Edward D. , Winokur Andrew
Published: December 2004

The distribution of the beta1 (β1) and beta2 (β2) subtypes of the β‐adrenergic receptor was examined in rat and nondiseased control human tissue. The distribution of the β1 and β2 receptors was also examined in schizophrenic cases, with additional studies in schizophrenic suicide and nonschizophrenic suicide cases. Scatchard analysis of the binding of [125I]iodopindolol (IPIN) to cortical membranes showed a similar Kd in human (177 pM) and rat (161 pM), but a lower maximum binding site (Bmax) in the human tissue (18.7 fmol/mg protein and 55.6 fmol/mg protein). For the autoradiographic studies [125I]IPIN was used to visualize both subtypes (total) or was displaced with the selective β1‐receptor antagonist ICI‐89,406 to visualize β2 sites, or with the selective β2‐receptor antagonist ICI‐118,551 to visualize β1 sites. Important differences in the regional distribution of the two subtypes of the β‐adrenergic receptors were noted between rat and human. In the nucleus accumbens and ventral putamen (ventral striatum), a patchy distribution of β1 receptors was observed that was not evident in the rat. These patches were aligned with markers of the matrix compartment of the striatum. The schizophrenic cases showed significant increases in the labeling of the β1‐receptor patches with [125I]IPIN. In contrast to the frontal cortex of the nondisease controls, the parietal and temporal cortex showed a high ratio of β1 to β2 receptors and a highly laminar organization of the subtypes. [125I]IPIN binding to β1 receptors was highest in the external laminae with the reverse gradient for the β2 subtype. The medial temporal cortex displayed an alteration in the ratio of the 2 subtypes of the β‐adrenergic receptor, with the parahippocampus and hippocampus of the human, in contrast to the rat brain, predominantly expressing the β2 receptor. Moreover, there were consistently higher densities of β2 receptors in the hippocampus of the right hemisphere than the left hemisphere of the nondisease controls. There was not a left and right hemispheric asymmetry of β2 receptors in the hippocampus of elderly schizophrenics or in young schizophrenics who committed suicide. The asymmetry was evident in nonschizophrenic suicides, suggesting that the lack of asymmetry in the hippocampus of schizophrenics is evident early in the disease process. Thus limbic structures show alterations in the patterning of β1 and β2 receptors in the schizophrenic cases.
Pharamacological characterization of excitaory synaptic potentials in rat basolateral amygdaloid neurons
Authors: Gean Po‐Wu , Chang Fang‐Chia
Published: December 2004

The pharmacological properties of synaptic responses in rat basolateral amygdaloid (BLA) neurons were studied using intracellular recording techniques. Three distinct types of synaptic potential were evoked by stimulation of the adjacent ventral endopyriform nucleus: (1) a fast excitatory postsynaptic potential (f‐EPSP);(2) a late EPSP (1‐EPSP) following the f‐EPSP; and (3) a multiphasic hyperpolarization following the initial depolarizing potential. Superfusion of 6‐cyano‐7‐nitroquinoxaline‐2, 3‐dione (CNQX), a selective non‐N‐methyl‐D‐aspartate (non‐NMDA) receptor antagonist, blocked the f‐EPSP in a concentration‐dependent manner. The ED50 for this effect was around 4 m̈M. In the presence of CNQX, however, a small depolarizing potential remained. This residual depolarizing component was markedly enhanced on removing Mg++ from the perfusing medium and could subsequently be abolished by DL‐2‐amino‐5‐ phosphonovaleate (DL‐APV, 50 m̈M) indicating its mediation via NMDA receptor‐coupled ionophore. The l‐EPSP was reversibly blocked by DL‐APV. These results suggest that the pyriform cortex‐amygdala pathway is mediated through excitatory amino acids acting on non‐NMDA as well as NMDA receptors located on the BLA neurons.
Neonatal 6‐OHDA Lesions differentially affect striatal D1 and D2 receptors
Authors: Neal Bethany S. , Joyce Jeffrey N.
Published: December 2004

Lesions to the dopamine (DA) system in early postnatal development have different behavioral consequences compared to lesions made in adulthood. Intrastriatal injections of the neurotoxin 6‐hydroxydopamine (6‐OHDA) on the day of birth (PO) or postnatal day 1 (P1) produce a selective supersensitivity to D1 receptor agonists and a subsensitivity to D1 antagonists (Neal and Joyce, 1991a). In this paper, we describe the long‐term effects of early DA loss on DA receptor regulation. Pups received bilateral intrastriatal injections of the neurotoxin 6‐OHDA (4 m̈g per striatum) on PO or P1. Adult rats were killed at 90 days of age and the brains were processed for quantitative autoradiography (QAR) or tyrosine hydroxylase (TH) immunocytochemistry. Cohorts were tested for the behavioral responses to the selective D1 receptor agonist SKF38393 (10 mg/kg). Neonatally lesioned rats exhibited increases in abnormal perioral movements in response to D1 receptor stimulation. There was a heterogenous and patchy loss (40–50%) of [3H]mazindol binding to high‐affinity DA uptake sites (a marker of DA terminal density) and a similar loss of TH‐like immunoreactivity within the striata of the neonatally lesioned rats. There was also a reduction in the number of ð‐opioid receptor patches (labelled with [3H] naloxone), a marker for the striatal patch compartment, and a similar patchy loss of D1 binding sites (labelled with [3H]SCH23390). The binding of [3H]spiroperidol to D2 sites was not altered. This is in contrast to the changes observed following adult 6‐OHDA lesions, wherein there is a significant increase in the number of D2 binding sites (Joyce, 1991a, b). The results are discussed with respect to the behavioral consequences of neonatal lesions and the differences between neonatal and adult lesions. © 1992 Wiley‐Liss, Inc.
Membrane‐Bound and solubilized brain 5HT3 receptors: Improved radioligand binding assays using bovine area postrema or rat cortex and the radioligands 3H‐Gr65630C, 3H‐BRl43694, and 3H‐LY278584
Authors: Miller Keith , Weisberg Ellen , Fletcher Paul W. , Teitler Milt
Published: December 2004

Bovine area postrema tissue was used as a convenient source of tissue for studies of brain 5HT3 receptor density was determined to be 97 ± 5 fmol/mg and 124 ± 10 fmol/mg of protein using the commercially available 5HT3 radioligands, 3H‐GR65630 and 3H‐BRL43694, respectively. The equilibrium dissociation constants (KD) for 3H‐GR65630 and 3H‐BRL43694 were 0.5 ± 0.1 nM and 1.7 ± 0.3 nM, respectively. The affinities of a series of drugs for the 5HT3 receptor using the two radioligands were essentially identical; the Ki values and order of affinities of agonists and antagonists were very similar to data published in studies on radiolabeling of 5HT3 receptors in rat brain. 3H‐LY278584 also labeled 5HT3 receptors in bovine area postrema homogenates with a KD of 3.1 ± 0.1 nM and a Bmax of 84 ± 6 fmol/mg. In rat cortical homogenates, 3H‐LY278584 produced the most reliable specific signal of 72%, with a KD of 2.6 ± 0.3 nM and a Bmax value of 10.5 ± 1 fmol/mg. At 1 nM, 3H‐GR65630 or 3H‐BRL43694 specific binding represented 28 and 50% of total radioligand binding, respectively. These data in bovine and rat brain tissues indicate that bovine area postrema can be used with 3H‐GR65630, 3H‐BRL43694, or 3H‐LY278584 can be used for studies on the regulation and/or the molecular properties of 5HT3 receptors in rat cortical homogenates. 5HT3 receptors were solubilized from bovine area postrema and characterized using 3H‐GR65630. Gel filtration chromatography revealed a specific binding peak with a MW of approximately 600 kDa. A 10‐fold enrichment of the binding activity was achieved using wheat germ agglutinin followed by application of N‐acetyl‐glucosamine to elute the glycosylated proteins, indicating the receptor is a glycosylated protein. © Wiley‐Liss, Inc.
Synaptic organization of the serotoninergic input to the superficial gray layer of the hamster's superior colliculus
Authors: Arce Erick A. , Bennett‐Clarke Carol A. , Mooney Richard D. , Rhoades Robert W.
Published: December 2004

Immunocytochemistry with an antibody directed against a serotonin (5‐HT)‐bovine serum albumin (BSA) conjugate was combined with electorn microscopy and serial section analysis to examine the synaptic organization of the serotoninergic projection to the stratum griseum superficale (SGS) of the hamster's superior colliculus (SC). Most of the 5‐HT‐immunoreactive swellings in the hamster's SC did not make conventional synaptic contacts. Examination of 250 such swellings in single thin sections revealed only 11 synapses. Assessment of an additional 50 swellings followed through serial sections showed that only 6% (N = 3) of these varicosities made conventional synaptic contacts. All the synapses made by serotoninergic axons were with the dendrites of SC neurons. These results demonstrate that the inhibitory effects of 5‐HT on SC neurons in hamster are mainly mediated by the nonsynaptic release of this indoleamine. © Wiley‐Liss, Inc.
Feedback inhibition of met‐enkephalin release from the rat spinal cord in vivo
Authors: Collin E. , Bourgoin S. , Mantelet S. , Hamon M. , Cesselin F.
Published: December 2004

The possible exitence of a feedback control by endogenous opioids of the spinal release of met‐enkephalin‐like material was assessed in vivo, in halothane‐anesthetized rats whose intrathecal space was continuously perfused with an artifical cerebrospinal fluid supplemented with various opioid‐related drugs. Both the intrathecal perfusion of the μ agonist D‐Ala2‐D‐MePhe4‐Gly‐ol5‐enkephalin (DAGO) (10 μM) and the ∂ agonist Tyr‐D‐Thr‐Gly‐Phe‐Leu‐Thr (DTLET) (10 μM) produced a significant inhibition of the spinal outflow of met‐enkephalin‐like material. The effect of DAGO, but not that of DTLET, could be prevented by naloxone (10 μM), and, conversely, the effect of DLTET, but not that of DAGO, was no longer observed in the presence of naltrindole (10 μM). Therfore naloxone and naltrindole acted as potent and selective μ and ∂ antagonists, respectively, when perfused at 10 μM in the intrathecal space of halothane‐anesthetized rats. As expected from the lack of a tonic opioid control of spinal enkephalinergic neurones, neither naloxone nor naltrindole alone affected the spontaneous outflow of metenkephalin‐like material. However, naltrindole, but not naloxone, markedly increased the spinal overflow of met‐enkephalin‐like material due to intrathecal administration of either porcine calcitonin (10 μM) or the peptidase inhibitors thiorphan (10 μM) plus bestatin (20 μM). These data suggest that μ, but not ð, receptors are involved in a phasic opioid inhibitory control of the release of met‐enkephalin‐like material in the rat spinal cord. This control would be functional only when the concentration of extracellular endogenous opioid(s) reaches a critical level, as probably occurring in pain‐suffering subjects. ð Opioid antagonists acting selectively on the receptors involved in this feedback control might constitute a new class of analgesic drugs. © 1992 Wiley‐Liss, Inc.
Chronic cocaine enhances serotonin autoregulation and serotonin uptake binding
Authors: Cunningham Kathryn A. , Paris Joseph M. , Goeders Nick E.
Published: December 2004

Repeated cocaine intoxication can result in the development of behavioral sensitization in animals and psychosis in humans, phenomena that have been associated with alterations in dopamine (DA) function. Using electrophysiologic and autoradiographic techniques, modifications of central serotonin (5‐hydroxytryptamine; 5‐HT) systems were investigated in rats treated with a regimen of cocaine administration that produced behavioral sensitization. The inhibitory response of single 5‐HT neurons in the dorsal raphe (DR) to (−)‐cocaine, the 5‐HT uptake inhibitor fluoxetine or the 5‐HT1A agonist 8‐hydroxy‐2‐[di‐N‐propylamino]tetralin (8‐OHDPAT) was significantly enhanced in cocaine‐treated rats. Furthermore, several brain areas that contain either cell bodies (DR) or terminals for 5‐HT (medial and sulcal prefrontal cortex, frontal cortex) showed cocaine‐induced elevations in [3H]imipramine‐labeled 5‐HT uptake sites, while [3H]‐8‐OHDPAT‐labeled 5‐HT1A receptors were decreased only in the central medial amygdala. These results suggest that modifications of autoregulatory mechanisms secondary to alterations of 5‐HT uptake processes may contribute to the development of cocaine sensitization. © Wiley‐Liss, Inc.
Effects of nigrostriatal lesions on the levels of messenger RNAs encoding two isoforms of glutamate decarboxylase in the globus pallidus and entopeduncular nucleus of the rat
Authors: Soghomonian Jean‐Jacques , Chesselet Marie‐Francoise
Published: December 2004

The neurotransmitter γ‐aminobutyric acid (GABA) is present in efferent neurons of the striatum and of the pallidum, one of the main striatal target areas. Dopaminergic nigrostriatal neurons play a critical role in the regulation of GABAergic neurotransmission in the striatum. In the present study, we investigated their role in the regulation of glutamate‐decarboxylase (GAD) mRNA expression in two divisions of the pallidum in rats: the globus pallidus and entopeduncular nucleus, equivalent to the external and internal pallidum, respectively, of primates. Dopaminergic neurons were lesioned by unilateral injections of 6‐hydroxydopamine (6‐OHDA) in the substantia nigra of adult rats. Two or 3 weeks after the lesion, frontal cryostat‐cut sections of the brain were processed for in situ hybridization histochemistry with 35S‐labeled RNA probes synthesized from cDNAs encoding two distinct isoforms of GAD of respective molecular weight 67,000 (GAD67) and 65,000 (GAD65). The number of labeled cells was determined, and intensity of labeling in individual cells was analyzed by computerized image analysis on emulsion radioautographs.

In the globus pallidus, the number of labeled neurons and intensity of labeling per cell were increased on the side ipsilateral to the lesion as compared with control rats in sections hybridized with the GAD67 RNA probe. No changes were detected on the side contralateral to the lesion or in the levels of labeling for GAD65 mRNA. Confirming previous data, the level of labeling for GAD65 mRNA was much higher than for GAD67 mRNA in the entopeduncular nucleus of control rats. In rats with a 6‐OHDA lesion, labeling for both GAD67 and GAD65 mRNAs was decreased on the side contralateral, but not ipsilateral, to the lesion, as compared with control rats.

The results show that lesions of the nigrostriatal pathway in rats affect the levels of mRNAs encoding two distinct isoforms of GAD in neurons of the globus pallidus and entopeduncular nucleus differently. In addition, results in the entopeduncular nucleus further support a bilateral effect of unilateral dopaminergic lesions. © 1992 Wiley‐Liss, Inc.
[123/125I]RTI‐55, an in vivo label for the serotonin transporter
Authors: Scheffel Ursula , Dannals Robert F. , Cline Elizabeth J. , Ricaurte George A. , Carroll F. Ivy , Abraham Philip , Lewin Anita H. , Kuhar Michael J.
Published: December 2004

[123I]RTI‐55, an iodinated derivative of the cocaine analog 3β‐phenyltropane‐2β‐carboxylic acid methyl ester, was evaluated as an agent for in vivo labeling of the serotonin transporter. Labeling of the precursor of RTI‐55 with I‐123 was efficient and yielded a high specific activity product. After intravenous injection of [123I]RTI‐55 into rats, the tracer accumulated in regions with high densities of serotonin and dopamine uptake sites. The distribution of [123I]RTI‐55 binding in areas rich in serotonin uptake sites correlated with [3H]serotonin uptake measured in vitro in the same regions. Specific [123I]RTI‐55 binding to serotonin uptake sites was inhibited by paroxetine but not by GBR 12,909. Treatment of rats with neurotoxic doses of fenfluramine caused decreases of 66% (in the hypothalamus) to 83% (in the superior colliculi) of specific [125I]RTI‐55 binding in all areas except in the striatum and the olfactory tubercles (regions rich in dopamine transporters). These results indicate that [123/125I]RTI‐55 binds, although not selectively, to the serotonin transporter in vivo. Furthermore, they suggest that [123I]RTI‐55 holds promise as a SPECT imaging agent for the study of the serotonin transporter in humans in health and disease. Published Wiley‐Liss, Inc.
Topographic nonoverlapping distribution of D1 and D2 dopamine receptors in the amygdaloid nuclear complex of the rat brain
Authors: Scibilia Richard J. , Lachowicz Jean E. , Kilts Clinton D.
Published: December 2004

The distribution of D1 and D2 dopamine (DA) receptors in the nuclei and subnuclear zones of the rat amygdaloid complex was mapped using quantitative light microscopic autoradiography. [125I]iodosulpiride and [125I]SCH 23982 (in the presence of 50 nM ketanserin) were used to label D2 and D1 DA receptors, respectively. The DA receptor subtypes exhibited a topographic, nonoverlapping distribution which generally conformed to the cytoarchitectonic boundaries of the component nuclei and subnuclear zones of the amygdaloid complex. The highest density of [125I]iodosulpiride binding sites was observed in the main intercalated cell group and the central amygdaloid nucleus where a medial to lateral gradient of binding sites was localized to its subnuclear zones. [125I]SCH 23982 binding sites were localized in the main intercalated cell group and the basolateral amygdaloid nucleus with a uniform low density in the central nucleus. The functional topography of mesoamygdaloid DA neurons may therefore be mediated, in part, at the level of DA receptor subtypes. The pattern of distribution of [125I]iodosulpiride binding sites in subdivisions of the central amygdaloid nucleus and bed nucleus of the stria terminalis suggests that the functions of the “extended amygdala,” a major system of the functional organization of the basal forebrain, may be regulated by DA afferents at multiple key sites of D2 receptor action. © Wiley‐Liss, Inc.
MK‐801 protection against methamphetamine‐induced striatal dopamine terminal injury is associated with attenuated dopamine overflow
Authors: Weihmuller Fredric B. , O'Dell Steven J. , Marshall John F.
Published: December 2004

Repeated administrations of methamphetamine (m‐AMPH) produce high extracellular levels of dopamine (DA) and subsequent striatal DA terminal damage. Pharmacological blockade of N‐methyl‐D‐aspartate (NMDA) receptors has been shown previously to prevent m‐AMPH‐induced striatal DA terminal injury, but the mechanism for this protection is unclear. In the present study, in vivo microdialysis was used to determine the effects of blockade of NMDA receptors with the noncompetitive antagonist MK‐801 on m‐AMPH‐induced striatal DA overflow. Four injections of MK‐801 (0.5 mg/kg, ip) alone did not significantly change extracellular striatal DA concentrations from pretreatment values. Four treatments with m‐AMPH (4.0 mg/kg, sc at 2‐hr intervals) increased striatal DA overflow, and the overflow was particularly extensive following the fourth injection. This m‐AMPH regimen produced a 40% reduction in striatal DA tissue content 1 week later. Treatment with MK‐801 15 min before each of the four m‐AMPH injections or prior to only the last two m‐AMPH administrations attenuated the m‐AMPH‐induced increase in striatal DA overflow and protected completely against striatal DA depletions. Other MK‐801 treatment regimens less effectively reduced the m‐AMPH‐induced striatal DA efflux and were ineffective in protecting against striatal DA depletions. Linear regression analysis indicated that cumulative DA overflow was strongly predictive (r = −.68) of striatal DA tissue levels measured one week later. These findings suggest that the extensive DA overflow seen during a neurotoxic regimen of m‐AMPH is a crucial component of the subsequent neurotoxicity. Blockade of NMDA receptors with MK‐801 apparently prevents damage to DA neurons by attenuating this stimulant‐induced DA overflow. © Wiley‐Liss, Inc.
Regional norepinephrine response to amphetamine using dialysis: Comparison with caudate dopamine
Authors: Kuczenski Ronald , Segal David S.
Published: December 2004

The response of extracellular norepinephrine to the acute administration of amphetamine was assessed, using dialysis, in prefrontal cortex and hippocampus in awake, behaving rats. Norepinephrine exhibited a pronounced and rapid dose‐ and time‐dependent increase in response to 0.5 and 2.5 mg/kg amphetamine, which corresponded closely to the time course of the behavioral profile. These results are consistent in with a possible role for norepinephrine in the behavioral response to amphetamine. © Wiley‐Liss, Inc.
Evidence for GABAergic interneurons in the red nucleus of the painted turtle
Authors: Keifer Joyce , Vyas Devhuti , Houk James C. , Berrebi Albert S. , Mugnaini Enrico
Published: December 2004

Immunocytochemical and electrophysiological evidence supporting the presence of GABAergic interneurons in the turtle red nucleus is presented. Injections of HRP into the spinal cord produced labeling of large neurons in the contralateral red nucleus. The peroxidase‐antiperoxidase (PAP) method revealed smaller cells immunoreactive to an antibody against glutamate decarboxylase (GAD), the synthetic enzyme for the inhibitory neurotransmitter GABA, that were interspersed among larger immunonegative neurons. Similar small neurons were densely immunostained by antibodies to GABA‐glutaraldehyde conjugates obtained from different sources and applied according to pre‐embedding and postembedding protocols. Rubrospinal neurons retrogradely labeled with HRP measured 16 and 27 μm in mean minor and major cell body diameters, while GABA‐like immunopositive neurons situated within the red nucleus measured 7 and 13 μm. There was very little overlap in soma size between the two cell populations. Therefore, we suggest that the GAD‐ and GABA‐positive neurons may be local inhibitory interneurons. This nition is further supported by observations of pre‐embedding immunostaining for GAD and postembedding immunostaining for GABA showing that the turtle red nucleus is amply innervated by immunoreactive axon terminals. These puncta are closely apposed to cell bodies and dendrites of both immunonegative large neurons and immunopositive small neurons. Moreover, immunogold staining at the electron microscopic level demonstrated that GABA‐like immunoreactive axon terminals with pleomorphic synaptic vesicles formed symmetric synapses with cell bodies and dendrites of the two types of red nucleus cells. These ultrastructural features are commonly assumed to indicate inhibitory synapses. A moderately labeled bouton with round vesicles and asymmetric synapses was also observed. In addition, the two types of red nucleus neurons received asymmetric axosomatic and axodendritic synapses with GABA‐negative boutons provided with round vesicles, features usually associated with excitatory functions. To obtain electrophysiological evidence for inhibition, intracellular recordings from red nucleus neurons were conducted using an in vitro brainstem‐cerebellum preparation from the turtle. Small, spontaneous IPSPs were recorded from 7 out of 14 red nucleus cells studied.

These morphological and physiological results provide strong support for concluding that the turtle red nucleus, like its mammalian counterpart, contains GABAergic inhibitory interneurons. While we have not indentified the main source of input to these interneurons, in view of the scarce development of the reptilian cerebral cortex, this input is unlikely to come from the motor cortex as it does in mammals. Current concepts of the role of these interneurons in motor control must take into account the presence of this interneuronal system that phylogenetically precedes the emergence of the sensorimotor cortex. © Wiley‐Liss, Inc.
ATP‐Sensitive K+ channels mediate an IPSP in dorsal horn neurones elicited by sensory stimulation
Authors: Salter Michael W. , De Koninck Yves , Henry James L.
Published: December 2004

Nociceptive dorsal horn neurones, which are involved in the processing of pain‐related information, are inhibited by input from vibration‐sensitive, large diameter primary sensory fibres (Wall and Cronly‐Dillon, 1960; Salter and Henry, 1990a, b). We have reported previously that the inhibition of spinal nociceptive neurones by vibration is mediated by adenosine acting through P1‐purinergic receptors (Salter and Henry, 1987). In a number of different types of cell, adenosine is known to activate K+ currents (Gerber et al., 1989; Greene and Haas, 1985; Proctor and Dunwiddie, 1987; Segal, 1982; Trussell and Jackson, 1987) and we have recently found that the adenosine‐mediated inhibition of nociceptive neurones by vibration is the result of an inhibitory postsynaptic potential (IPSP), which is, indeed, caused by a K+ conductance (De Koninck and Henry, 1988, 1992). It has been reported that adenosine‐activated K+ channels in cardiac muscle cells are the ATP‐sensitive K+ channels (Kirsch et al., 1990). Therefore, we questioned whether these channels might mediate the purinergic IPSP we have observed in nociceptive dorsal horn neurones. We report here that glibenclamide, a blocker of ATP‐sensitive K+ channels (Ashcroft, 1988; Schmid Antomarchi et al., 1987a, b), blocks the inhibition of nociceptive neurones by vibratory stimulation when this compound is administered locally by iontophoresis or systemically by intravenous injection. In addition, direct intracellular injection of ATP was found to block the IPSP evoked by vibratory stimulation. These data indicate that the purinergic IPSP in nociceptive spinal neurones is mediated via ATP‐sensitive K+ channels. As inhibition of nociceptive dorsal horn neurones may be the physiological basis for the analgesia produced by vibratory stimulation in humans (Bini et al., 1984; Lundeberg, 1984; Lundeberg et al., 1984; Ottoson et al., 1981), we propose that activation of ATP‐sensitive K+ channels in these neurones may mediate the analgesia produced by vibration. © Wiley‐Liss, Inc.
Oscillation of interspike interval length in substantia nigra dopamine neurons: Effects of nicotine and the dopaminergic D2 agonist LY 163502 on electrophysiological activity
Authors: Carlson Joanne H. , Foote Stephen L.
Published: December 2004

The rates and patterns of discharge activity exhibited by 16 spontaneously active substantia nigra pars compacta dopamine neurons were studied in halothane‐anesthetized rats using three types of quantitative measures: (1) mean discharge rates, (2) population characteristics of interspike interval samples, and (3) interspike interval time‐series measures which were used to examine patterns in the ordering of interspike intervals. The mean discharge rate of these 16 cells was 2.9 ± 0.3 spikes/sec, and each cell was classified as bursting (25% of the cells) or non‐bursting (75%). The distribution of interspike intervals of bursting cells was typically positively skewed and sometimes bimodal, while the interspike intervals of non‐bursting neurons were more normally distributed. Time‐series analyses (raw time‐series plots, return maps, and phase portraits) revealed a substantial oscillatory tendency in the magnitudes of consecutive interspike intervals in these neurons under baseline conditions: Successive interspike intervals tended to alternate between short and long durations, although short bursts often occurred. Under baseline conditions, these cells exhibited both multispike bursts and consecutive long intervals less frequently than would have been predicted by chance ordering of the interspike intervals. These results imply that there are mechanisms acting to reduce the probability of these types of events.

Locally infused nicotine enhanced discharge rates in these neurons. Burst firing increased in four neurons, while five neurons did not show any change in burst firing. LY 163502 induced significant decreases in both discharge rate and bursting activity in all cells tested. The variation coefficient, skew, and kurtosis of the interspike interval distributions were not consistently altered by either drug. The local infusion of either nicotine or LY 163502 decreased the oscillatory phenomenon seen in the baseline condition. Neither the nicotine or LY 163502 time‐series data exhibited a larger proportion of long‐short and short‐long pairs (relative to the median interval) than would be expected by chance.

It is hypothesized that these neurons have intrinsic mechanisms, made manifest under anesthesia, which induce oscillations in interspike interval length. The oscillatory effect of these mechanisms can be overridden by tonic increases in either excitatory or inhibitory tone. © Wiley‐Liss, Inc.