Notes: Inherited congenital myoclonus (ICM) of Poll Hereford cattle is a neurological disease in which there are severe alterations in spinal cord glycine-mediated neurotransmission. There is a specific and marked decrease, or defect, in glycine receptors and a significant increase in neuronal (synaptosomal) glycine uptake. Here we have examined the characteristics of the cerebral γ-aminobutyric acid (GABA) receptor complex, and demonstrate that the malfunction of the spinal cord inhibitory system is accompanied by a change in the major inhibitory system in the cerebral cortex. In synaptic membrane preparations from ICM calves, both high- and low-affinity binding sites for the GABA agonist [3H]muscimol were found (KD= 9.3 ± 1.5 and 227 ± 41 nM, respectively), whereas only the high-affinity site was detectable in controls (KD= 14.0 ± 3.1 nM). The density and affinity of benzodiazepine agonist binding sites labelled by [3H]diazepam were unchanged, but there was an increase in GABA-stimulated benzodiazepine binding. The affinity for t-[3H]butylbicyclo-o-benzoate, a ligand that binds to the GABA-activated chloride channel, was significantly increased in ICM brain membranes (KD= 148 ± 14 nM) compared with controls (KD= 245 ± 33 nM). Muscimol-stimulated 36Cl- uptake was 12% greater in microsacs prepared from ICM calf cerebral cortex, and the uptake was more sensitive to block by the GABA antagonist picrotoxin. The results show that the characteristics of the GABA receptor complex in ICM calf cortex differ from those in cortex from unaffected calves, a difference that is particularly apparent for the low-affinity, physiologically relevant GABA receptors. Study of the GABA receptor complex in ICM calves may provide a greater insight into the interactions of the various binding sites on the GABA-benzodiazepine receptor protein molecule.

Notes: Abstract: Cerebral cortex tissue was obtained at autopsy from neonatal Poll Hereford calves with clinically confirmed maple syrup urine disease (MSUD), neonatal Holstein-Friesian calves with clinically confirmed citrullinemia, and matched controls. From this, synaptosomes were prepared for studies of neurotransmitter amino acid uptake and stimulus-induced release, and synaptic plasma membranes were obtained for studies of associated postsynaptic receptor binding sites. As well as having abnormal brain tissue concentrations of the pathognomic plasma amino acids (markedly increased levels of the branched-chain compounds valine, isoleucine, and leucine in MSUD; marked elevation of citrulline levels in citrullinemia), both groups of diseased animals showed reduced, brain tissue concentrations of each of the transmitter amino acids glutamate, aspartate, and γ-aminobutyric acid (GABA). Nontransmitter amino acids were generally unaffected in either disease. Citrullinemic calves showed a marked increase in brain glutamine concentration; in calves with MSUD, the glutamine concentration was raised, but to a much lesser extent. The Na+-dependent synaptosomal uptake of both glutamate and GABA was markedly reduced (to 〈50% of control values in both cases) in citrullinemic calves but was unaltered in calves with MSUD. Whereas synaptosomes from normal calves showed the expected stimulus-coupled release of transmitter amino acids, especially glutamate and aspartate, and no response to stimulus of nontransmitter amino acids, there was no increased release of transmitter amino acids in response to depolarization in synaptosomes from citrullinemic calves. This was in part because the extracellular concentrations of these compounds in citrullinemic control incubations were already high, especially for glutamate—basal extrasynaptosomal glutamate concentrations were some 20-fold higher than those found with synaptosomes from normal calves—so that further stimuluscoupled enhancement was not possible. Calves with MSUD showed a marked loss in number of postsynaptic GABAA receptors (to ∼-50% of normal values), as assessed from [3H]diazepam binding studies. In contrast, there was no loss of this receptor site in citrullinemic calves. Calves with citrullinemia showed a marked reduction in the affinity and density of postsynaptic glutamate N-methyl-D-aspartate receptors as assessed from [3H]MK-801 binding studies. In contrast, calves with MSUD showed no change in this parameter. These studies show that two major recessively inherited diseases of cattle have similar, but distinct, neurochemical pathologies. The MSUD encephalopathy appears to be driven by a diminution of GABA-mediated inhibitory neurotransmission, whereas in citrullinemia the equivalent proconvulsive state may be driven by a relative increase in glutamate-mediated excitatory activity.

Notes: Cortical spreading depression (CSD) is characterized by slowly propagating waves of neuronal/astrocytic depolarization and metabolic changes, followed by a period of quiescent neuronal and electroencephalographic activity. CSD acts as a preconditioning stimulus in brain, reducing cell death when elicited up to several days prior to an ischemic insult. Precise mechanisms associated with this neuroprotection are not known, although CSD increases the expression of a number of potentially neuroprotective genes/proteins. The nitric oxide (NO) system may be of particular importance, as it is acutely activated and chronically up-regulated in cerebral cortex by CSD, and NO can ameliorate and exacerbate cell death under different conditions. Several molecules have recently been identified that modulate the production and/or cellular actions of NO, but it is not known whether their expression is altered by CSD. Therefore, the present study examined the effect of CSD on the spatiotemporal expression of PIN, CAPON, PSD-95, Mn-SOD and Cu/Zn-SOD mRNA in the rat brain. In situ hybridization using specific [35S]-labelled oligonucleotides revealed that levels of PIN mRNA were significantly increased in the cortex and claustrum (∼30–180%; p ≤ 0.01) after 6 h and 1 and 2 days, but were again equivalent to contralateral (control) cortical values at 7, 14 and 28 days. CAPON mRNA levels were increased (∼30–180%; p ≤ 0.05) in the ipsilateral cortical hemisphere at 6 h and 2 days post treatment, but not at the other times examined. In contrast, levels of PSD-95, Mn- and Cu/Zn-SOD mRNA were not altered at any time after CSD. These results suggest that following CSD, nNOS activity and NO levels may be tightly regulated by both transcriptional and translational alterations in a range of nNOS adaptor proteins, which may contribute to CSD-induced neuroprotection against subsequent ischemia.

Notes: Abstract: Galanin has been ascribed several physiological roles that are thought to be mediated via multiple galanin receptors. Recently, two galanin receptors—galanin receptor-1 (GalR1) and galanin receptor-2 (GalR2)—have been cloned and characterized and shown to have differences in amino acid sequence, pharmacology, and second messenger signaling systems. Previous studies have demonstrated an up-regulation of galanin expression in damaged neurons of several different types. Using in situ hybridization histochemistry this study investigated whether adult cranial motor neurons express mRNAs encoding GalR1 and/or GalR2 and explored possible time-dependent changes in these transcripts following facial nerve injury. GalR2 mRNA levels were increased in the ipsilateral facial nucleus 3 (∼1.8-fold) and 7 days (∼3.7-fold) after unilateral facial nerve crush and had returned to levels equivalent to those in contralateral controls by 14–21 days. GalR1 mRNA was not detected in facial nuclei of naive, sham-operated, or operated rats but was present in adjacent reticular nuclei. Galanin mRNA levels were also increased eight- to 10-fold in the ipsilateral facial nucleus following nerve injury. These experiments confirm the putative importance of galanin signaling systems after nerve injury by demonstating a differential response of galanin receptor subtypes and suggest an important “autoreceptor” role for the GalR2 receptor in these processes.

Notes: Cholecystokinin (CCK) has been suggested to mediate satiety in a number of non-primate species via its peripheral actions as well as a possible central mechanism involving magnocellular and parvocellular oxytocin release. Quantitative in vitro autoradiography employing [125l]-Bolton-Hunter labelled CCK-8S ([125I]-CCK-8S) was used to examine the distribution and density of CCK receptors in sections of brain from normal rats and rats deprived of food, water or both food and water for 4 days. In food-deprived rats, specific [125I]-CCK-8S binding was reduced by 64 ± 5% in the hypothalamic supraoptic nucleus (SON) and by 44±13% in the paraventricular nucleus of the hypothalamus (PVN). In contrast, water deprivation increased binding of [125I]-CCK-8S by 128±15% in the SON and by 196 ± 24% in the PVN, while combined food and water deprivation produced smaller increases in both nuclei (30 ± 5% and 98 ± 26% in SON and PVN respectively). Changes in receptor density in the PVN appeared to be most prominent in the magnocellular (especially oxytocin-rich) subdivisions. None of the treatments employed produced changes in [125I]-CCK-8S binding in the ventromedial hypothalamic nucleus or the reticular thalamic nucleus. Both CCK-A and CCK-B receptor subtypes were visualized in the nucleus of the solitary tract and the area postrema of normal rats, but levels of binding to both of these subtypes were unaffected by the experimental treatments. These selective alterations demonstrate the plasticity of CCK receptors in the SON and PVN, and are probably associated with changes in the level of neurochemical activity of magnocellular oxytocinergic neurones in these areas. These results, together with reports of changes in the level of CCK synthesis in cells of the SON and PVN after hyperosmotic stimuli, suggest that CCK may act in an autocrine fashion on these neurones and that both CCK receptors and peptide levels are altered in the same direction following cellular activation or inhibition.

Notes: The distribution of messenger ribonucleic acid (mRNA) encoding preproneuropeptide Y (prepro-NPY) in the hypothalamus of rats subjected to food deprivation or dehydration has been investigated by quantitative in situ hybridization. Levels of prepro-NPY mRNA in the arcuate nucleus (ARC) were selectively increased by both treatments. The very high concentration of prepro-NPY mRNA seen following 96 h of food deprivation had returned towards control levels after 24 h of refeeding. Levels of preprogalanin (prepro-GAL) mRNA throughout the hypothalamus were essentially unaffected by both regimes. These results demonstrate that hypothalamic NPY gene expression is regulated by peripheral metabolic status (and osmolality), and confirm the key physiological role of NPY in controlling ingestive behaviour.

Notes: 1. Galanin peptide and galanin receptor-binding sites are known to be widely distributed within the central nervous system, particularly in the hypothalamus in the preoptic area, the paraventricular (PVN) and supraoptic (SON) nuclei and the arcuate nucleus/median eminence.2. The present brief review focuses on some recent studies of the regional and cellular localization of mRNA encoding galanin and two galanin receptor subtypes (GalR1 and GalR2) in the hypothalamus, regulation of galanin and/or galanin receptor expression in various nuclei by physiological stimuli, electrophysiological effects of galanin on hypothalamic neurons and the isolation and cloning of galanin-like peptide (GALP), a putative endogenous ligand for GalR2.3. In situ hybridization studies in rat brain have demonstrated an abundance of GalR1 mRNA in SON, magnocellular (m) and parvocellular (p) PVN and dorsomedial, ventromedial and arcuate nuclei. In contrast, GalR2 mRNA is enriched in pPVN, but not mPVN, and is not detected in SON. In addition, GalR2 mRNA is present in the dorsomedial nucleus and is enriched in the arcuate nucleus compared with GalR1 transcripts, with numerous labelled cells in all subdivisions.4. Neurons of the SON and PVN contain vasopressin and/or oxytocin, along with several other peptides, and the production and release of these hormones and peptides are modulated by various physiological stimuli. In relation to galanin systems, GalR1 and galanin expression is increased in magnocellular neurons by salt loading and is downregulated by lactation, consistent with an increased inhibition by galanin of vasopressin release following osmotic stimulation and a decreased inhibition of oxytocin release during lactation.5. Powerful inhibitory effects of galanin on the electrical (and secretory) activity of magnocellular neurons and complex presynaptic actions of galanin on the synaptic release of glutamate in the arcuate nucleus in vitro suggest an active role for multiple galanin receptor subtypes in the regulation of these hypothalamic systems in vivo.6. The recent isolation of a peptide from porcine hypothalamus (GALP-1–60) that is structurally related to galanin and appears to be selective for GalR2 over GalR1 and the subsequent cloning of GALP cDNA from pig, rat and humans should allow studies to help reveal the physiological role played by galanin receptor subtypes (especially GalR2) and their multiple ligands in the hypothalamus and other brain areas.

Notes: The peptide-hormone relaxin has well-established actions in male and female reproductive tracts, and has functional effects in circumventricular regions of brain involved in neurohormonal secretion. In the current study, we initially mapped the distribution of mRNA encoding the relaxin receptor − leucine-rich repeat-containing G-protein-coupled receptor 7 (LGR7)- and [33P]-human relaxin-binding sites in extra-hypothalamic sites of male Sprague–Dawley rats. The basolateral amygdala (BLA) expressed high levels of LGR7 mRNA and relaxin-binding sites and, although relaxin peptide was not detected in the BLA, several brain regions that send projections to the BLA were found to contain relaxin-expressing neurons. As it is well established that the BLA is involved in regulating the consolidation of memory for emotionally arousing experiences, we investigated whether activation of LGR7 in the BLA modulated memory consolidation for aversively motivated inhibitory avoidance training. Bilateral infusions of human relaxin (10–200 ng in 0.2 µL) into the BLA immediately after inhibitory avoidance training impaired 48-h retention performance in a dose-dependent manner. Delayed infusions of relaxin into the BLA 3 h after training were ineffective, indicating that the retention impairment was due to influences on memory consolidation. Post-training infusions of relaxin into the adjacent central amygdala, which is devoid of LGR7, did not impair retention. These findings suggest a novel function for endogenous relaxin–LGR7 signalling in rat brain involving regulation of memory consolidation.

Notes: Neocortex contains very few galanin neurons but receives a moderate galanin innervation from various subcortical loci. Recent data suggest that galanin helps regulate the tonic neuronal excitability of hippocampus and probably cerebral cortex but relatively little is known about the anatomy and functional regulation of cortical galanin systems. Therefore, we examined, in the rat, the effect of the intense but benign stimulus, cortical spreading depression (CSD), on the expression of galanin and galanin receptors (GalR1 and GalR2) in the neocortex and associated regions, revealing complex, multicellular responses. Thus, following acute, unilateral KCl-induced CSD, a delayed and transient induction (onset after 48 h, lasting ∼24 h) of galanin mRNA and peptide production occurred across the ipsilateral cerebral cortex in activated oligodendrocyte progenitor cells (OPCs), identified by specific NG2 proteoglycan immunostaining. An increase in GalR1 mRNA, immunoreactivity and receptor binding occurred in neurons within layers II and V of neocortex and in piriform cortex at 7–28 days after CSD, associated with a long-lasting depletion of galanin-positive nerve fibres in these regions. In contrast, GalR2 mRNA expression was largely unaltered after CSD. Additional novel findings in normal, adult brain were the detection of galanin mRNA and immunoreactivity in OPCs within the medial corpus callosum and in immature progenitor cells in the subventricular zone and rostral migratory stream. GalR1 and GalR2 mRNA was also present in these latter regions. These findings and the complex modulation of galanin and galanin receptors in multiple cell types (neurons/OPCs) following acute cortical activation/depression further demonstrate the potential plasticity of neuronal and non-neuronal galanin systems under physiological and pathological conditions and strongly suggest additional functions for this pleiotropic peptide in mammalian brain.

Notes: The present study employed 35S-labelled oligonucleotides and in situ hybridization to examine the distribution in the developing rat brain of mRNA encoding two galanin receptor subtypes, i.e. Gal-R1 and Gal-R2. Gal-R1 and/or Gal-R2 mRNA was detected at embryonic day (E) 20 and from postnatal day (P) 0–70. Gal-R1 mRNA was highly expressed in olfactory regions, ventral hippocampal CA fields, dorsomedial thalamic areas and many hypothalamic nuclei at all ages studied. In adult brain, Gal-R2 mRNA was most abundant in the dentate gyrus, anterior and posterior hypothalamus, raphe and spinal trigeminal nuclei, and in the dorsal motor nucleus of the vagus. At P0–P7, Gal-R2 mRNA was more widely distributed and abundant than at other ages, with highest levels of expression detected throughout the neocortex and thalamus. Thus, Gal-R2 transcripts had a more restricted distribution than Gal-R1 and were differentially abundant at different ages, while the distribution and relative abundance of Gal-R1 mRNA did not alter substantially during postnatal development. In general, Gal-R1 and -R2 mRNAs were localized in regions previously shown to contain [125I]-galanin binding sites and galanin-positive terminals in adult brain. Galanin-immunostaining was assessed in postnatal brain to determine whether peptide innervation correlated with observed transient receptor expression, but was not particularly enriched in Gal-R2 mRNA-positive areas of P4 or P7 brain. These results, together with earlier findings [e.g. Burazin, T. C. D. & Gundlach, A. L. (1998) J. Neurochem., 71, 879–882], suggest that Gal-R1 receptors have a broad role in normal synaptic transmission, while Gal-R2 receptors, in addition to a similar role in particular pathways, may be involved in processes prominent during the establishment and maturation of synaptic connections in developing brain and during neural damage and repair in the mature nervous system.