Phenylglycines can Evoke Quisqualate-primed Depolarizations in Rat Cingulate Cortex: An Effect Associated with [3H]DL-AP4 Uptake

Depolarization could be evoked in slices of rat cingulate cortex by the normally non-excitatory compound L-2-amino-4-phosphonobutyrate ( l -AP4) if the slices had been sensitized by exposure to quisqualate. The magnitude of the response to l -AP4 was dependent on the concentrations of both l -AP4 and quisqualate and was inhibited by α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor antagonism. A series of phenylglycine analogues were capable of evoking similar dose-dependent depolarizations in the rat cingulate cortex following quisqualate sensitization, the most potent being ( S )-4-carboxy-3-hydroxyphenylglycine. If the superfusate collected during application of ( S )-4-carboxy-3-hydroxyphenylglycine to a quisqualate-sensitized slice was administered to a slice not previously exposed to quisqualate, a small depolarization was obtained. All the compounds shown to be capable of evoking the quisqualate-sensitized response showed affinity for the l -AP4 uptake site whilst having no affinity at ionotropic glutamate receptors and different profiles of activity at metabotropic glutamate receptors. None of the compounds was active at the metabotropic glutamate 4a receptor. There was a statistically significant correlation between a compound's effectiveness in inhibiting [³H]DL-AP4 uptake into rat cortical synaptosomes and its potency in evoking quisqualate-sensitized depolarization. It is concluded that this response may be the result of hetero-exchange between L-AP4 ligands and quisqualate.     

Neurotrophic ACTH4–9 analogue therapy normalizes electroencephalographic alterations in chronic experimental allergic encephalomyelitis

Chronic experimental allergic encephalomyelitis (CEAE) is an established experimental model for multiple sclerosis (MS). The demyelinating lesions in the white matter of the central nervous system observed in CEAE and in MS are accompanied by various neurophysiological alterations. Among the best defined electrophysiological abnormalities are the changes in event-related potentials, in particular evoked potentials involving the spinal cord, i.e. motor and sensory evoked potentials. Less familiar are the changes observed in the electroencephalogram of CEAE-affected animals, which are also encountered in the human equivalent, MS. In the present experiment we evaluated the therapeutic value of a neurotrophic peptide treatment [H-Met(O₂)-Glu-His-Phe-d -Lys-Phe-OH, an ACTH_4–9 analogue] and its effect on the delayed flash visual evoked potentials (VEP) and power spectra of the electroencephalogram, during a 17-week follow-up of CEAE. CEAE animals treated with the neurotrophic peptide were protected against the development of neurological symptoms during the course of the demyelinating syndrome. VEPs of animals suffering from CEAE showed a delay of the latencies of the late components which was significantly counteracted by peptide treatment. The peak-to-peak amplitude of the VEP afterdischarge recorded from CEAE animals was significantly increased during the course of CEAE and correlated closely with the progression of the myelinopathy. Furthermore, CEAE animals showed an increase of electroencephalogram (EEG) beta activity of up to 500% as compared with the age-matched control group. This increase in beta power mainly consisted of a prevailing 20–21 Hz peak, a frequency that normally is not dominant in control EEG recordings of the rat during passive wakefulness. All these electrophysiological phenomena were absent in ACTH_4–9 analogue-treated animals. The present findings underscore the potential importance of a neurotrophic peptide treatment in the pharmacotherapy of central demyelinating syndromes, and possibly of MS.     

Autoradiographic Study of Binding and Internalization of a Luteinizing Hormone-Releasing Hormone Antagonist [D-Nal1, D-Cpa2, A-D-Trp3, D-Arg6, D-Ala10]LHRH by Rat Pituitary Gonadotrophs

The binding and intracellular pathway of the radioiodinated luteinizing hormone-releasing hormone (LHRH) antagonist [D-Nal¹, D-Cpa², D-Trp³, D-Arg⁶, D-Ala¹⁰]LHRH in pituitary gonadotrophs was studied as a function of time after iv injection of label into intact and castrated female rats. In semithin (1 |im) sections, silver grains were exclusively localized over about 10% of anterior pituitary cells in intact animals. In castrated animals, only the large castration cells were labeled. In control rats injected with both iodinated antagonist and an excess of unlabeled peptide, no significant labeling could be detected. At the ultrastructural level, the silver grains were exclusively localized in gonadotrophic cells. The time-course study showed that 30 min and 60 min after injection a high proportion of the silver grains was associated with the plasma membrane. Six h after injection, an appreciable proportion of the label was found over intracellular organelles, especially lysosomes and secretory granules. These results indicate that the potent LHRH antagonist used in the present experiments binds selectively to gonadotrophs and is subsequently internalized but at a much lower rate than that observed with LHRH agonists. This slow internalization of the LHRH antagonist might be related to the normal endocytic processes which occur independently of receptor activation.     

In Situ Hybridization of trkB and trkC Receptor mRNA in Rat Forebrain and Association with High-affinity Binding of [125I]BDNF, [125I]NT-4/5 and [125I]NT-3

The TrkB and TrkC receptor tyrosine kinases have been identified as high-affinity receptors for the neurotrophic factors brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) and NT-3 respectively. These receptor classes were identified and mapped by the in situ hybridization of antisense riboprobes complementary to portions of the intracellular (tyrosine kinase) or extracellular (ligand-binding) domains of trkB and trkC mRNA, and by the distribution of high-affinity [¹²⁵I]BDNF, [¹²⁵I]NT-4/5 and [¹²⁵I]NT-3 binding sites in adjacent rat brain sections. Both methods showed that TrkB and TrkC receptors are abundant and widely expressed throughout the brain. Kinase or extracellular domain trkC probes labelled neuronal somata in a qualitatively similar manner in virtually every major area of the forebrain. Neither trkC probe labelled non-neuronal cells except for elements within cerebral arteries and arterioles. The kinase domain trkB probe hybridized exclusively to neurons. Neurons expressing trkB were even more widely distributed than those expressing trkC. The extracellular domain trkB probe labelled neurons with the same relative distribution as the trkB kinase domain probe, but also hybridized extensively with non-neural cells, particularly astrocytes, ependyma and choroid epithelium cells. The distribution of [¹²⁵I]NT-3 binding sites generally resembled that of trkC hybridization, particularly in the neocortex, striatum and thalamus. [¹²⁵I]BDNF and [¹²⁵I]NT-4/5 binding sites were more widely distributed and denser than those for [¹²⁵I]NT-3, and resembled the trkB hybridization pattern. These patterns are consistent with the preferential binding in the brain of TrkC receptors by [¹²⁵I]NT-3 and of TrkB receptors by [¹²⁵I]BDNF and [¹²⁵I]NT-4/5. That the predominantly neuronal patterns of hybridization obtained with kinase and extracellular domain probes for trkC are qualitatively indistinguishable suggests that truncated and full-length forms of TrkC are expressed within extensively overlapping populations of neurons. In marked contrast to TrkC, expression of the full-length and truncated forms of TrkB appears to be largely segregated, being expressed principally on neurons and non-neuronal cells respectively. The abundant and widespread neuronal distribution of full-length, signal-transducing forms of TrkB and TrkC predict that their cognate ligands, BDNF, NT-4/5 and NT-3, may exert direct effects on a large proportion of neurons within the mature brain.     

Expression and Subcellular Localization of p42IP4/ Centaurin-α, a Brain-specific, High-affinity Receptor for lnositol 1,3,4,5-Tetrakisphosphate and Phosphatidylinositol 3,4,5-Trisphosphate in Rat Brain

Recently emerging evidence suggests important roles for inositol polyphosphates and inositol phospholipids in neuronal Ca²⁺ signalling, membrane vesicle trafficking and cytoskeletal rearrangement. A prerequisite for a detailed physiological characterization of the signalling of both potential second messengers inositol-(1,3,4,5)-tetrakisphosphate (InsP₄) and phosphatidylinositol-3,4,5-trisphosphate (PtdlnsP₃) in the nervous system is the precise cellular localization of their receptors. Based on the cDNA sequence of a recently cloned brain-specific receptor with high affinity for both InsP₄ and PtdlnsP₃ (InsP₄–PtdlnsP₃R), p42^IP4/centaurin-α, we localized the mRNA and the protein in rat brain. In situ hybridization revealed a widespread expression of the InsP₄–PtdlnsP₃R with prominent labelling in cerebellum, hippocampus, cortex and thalamus, which moreover is developmentally regulated. Using peptide-specific antibodies, the immunoreactivity was localized in the adult brain in the vast majority of neuronal cell types and probably also in some glial cells. Prominent immunoreactivity was found in axonal processes and in cell types characterized by extensive neurites. In the hypothalamus a subpopulation of parvocellular neurons in the peri- and paraventricular nuclei was most heavily labelled. This was confined by strong immunoreactivity in the lamina externa of the median eminence in close proximity to portal plexus blood vessels. Electron microscopy revealed that the InsP₄-PtdlnsP₃R was frequently associated with presynaptic vesicular structures. Further studies should identify the role of the InsP₄-PtdlnsP₃R in cellular neural processes.     

Distribution of [Leu,Pro]NPY-sensitive, BIBP3226-insensitive [I]PYY(3–36) binding sites in rat brain: possible relationship to Y5 NPY receptors

Recently, using molecular cloning approaches, three new neuropeptide Y (NPY)/peptide YY (PYY) receptors have been described in rodent brain, with pharmacological profiles that differ from the three previously described Y₁, Y₂ and Y₃ NPY receptors and the Y₄ pancreatic polypeptide- (PP-) preferring receptor. Two of these new receptors are spice variants and are called Y₅ receptors, whilst a third receptor has been called Y₆ and has been suggested to be expressed only in the mouse. In the absence of a totally selective Y₅ and/or Y₆ radioligands, we have examined [¹²⁵I]PYY(3–36) binding, which binds Y₂ and Y₅/Y₆ receptors, using homogenate assays and quantitative receptor autoradiography to study the distribution of the three newly discovered Y₅/Y₆ receptors by masking binding to Y₁ receptors with high concentrations of the non-peptidergic selective Y₁ antagonist, BIBP3226, and using either [Leu³¹,Pro³⁴]NPY or human PP to mask binding to Y₅ and Y₆ receptors, leaving binding to Y₂ receptors. Using this approach, [¹²⁵I]PYY(3–36) labels a small population of Y₁ receptors and a larger population of binding sites that are insensitive to BIBP3226, human PP and [Leu³¹,Pro³⁴]NPY, presumed to be Y₂ receptors. There was also [¹²⁵I]PYY(3–36) binding to sites sensitive to NPY, human PP and [Leu³¹,Pro³⁴]NPY, but insensitive to BIBP3226, located in the hypothalamus, amygdala, hippocampus and thalamus. As one of the recently cloned Y₅ receptors is synthesized in these regions, as shown by in-situ hybridization techniques, we suggest that the small population of [¹²⁵I]PYY(3–36) binding sites which are sensitive to human PP and [Leu³¹,Pro³⁴]NPY, but insensitive to BIBP3226, may represent binding to Y₅ receptors. We have been unable, however, to visualize a smaller population of Y₆ receptors which are labelled by [¹²⁵I]PYY₃–36 and sensitive to [Leu³¹,Pro³⁴]NPY, but not to BIBP3226 and human PP, confirming that the murine Y₆ receptor does not appear to be expressed in rat brain.