Johan Sandin

Nociceptin/Orphanin FQ Microinjected into Hippocampus Impairs Spatial Learning in Rats

The newly discovered peptide nociceptin/orphanin FQ has been found to increase reactivity to pain and to influence locomotor activity after intracerebroventricular administration. This study investigated the possible role of hippocampal nociceptin/orphanin FQ in spatial learning and in spontaneous locomotion. Male rats were trained in the Morris water task after microinjection of 10 nmol nociceptin/orphanin FQ or artificial cerebrospinal fluid (as control) into the CA3 region of the dorsal hippocampus. Nociceptin/orphanin FQ was found to severely impair spatial learning without interfering with swimming performance. Itrahippocampal injection of nociceptin/orphanin FQ markedly decreased exploratory locomotor activity including vertical movements (rearing). The data suggest that nociceptin/orphanin FQ is a potent modulator of synaptic plasticity within the hippocampus.

Analysis of the role of the 5-HT1B receptor in spatial and aversive learning in the rat.

The present study examined the role of the 5-HT1B receptor in learning and memory. The ability of the 5-HT1B receptor agonist anpirtoline and the selective 5-HT1B receptor antagonist NAS-181 to affect spatial learning in the water maze (WM) and aversive learning in the passive avoidance (PA) task were examined in the rat. Anpirtoline (0.1–1.0 mg/kg, s.c.) caused a dose-dependent impairment of learning and memory in both the WM and PA tasks. NAS-181 (1.0–10 mg/kg, s.c.) failed to alter performance of the WM task, but produced a dose-dependent (0.1–20 mg/kg) facilitation of PA retention. Furthermore, treatment with NAS-181 (10 mg/kg) fully blocked the impairment of the WM and PA performance caused by anpirtoline (1.0 mg/kg). In contrast, NAS-181 (3.0–10 mg/kg) did not attenuate the spatial learning deficit and the impairment of PA retention caused by scopolamine (0.1 mg/kg in WM task, 0.3 mg/kg in PA task, s.c.), a nonselective muscarinic antagonist. Moreover, a subthreshold dose of scopolamine (0.1 mg/kg) blocked the facilitation of PA retention induced by NAS-181 (1.0–10 mg/kg). In addition, the behavioral disturbances (eg thigmotaxic swimming and platform deflections) induced by anpirtoline and scopolamine were analyzed in the WM task and correlated with WM performance. These results indicate that: (1) 5-HT1B receptor stimulation and blockade result in opposite effects in two types of cognitive tasks in the rat, and that (2) the 5-HT1B antagonist NAS-181 can facilitate some aspects of cognitive function, most likely via an increase of cholinergic transmission. These results suggest that 5-HT1B receptor antagonists may have a potential in the treatment of cognitive deficits resulting from loss of cholinergic transmission.

Intraseptal muscarinic ligands and galanin: influence on hippocampal acetylcholine and cognition

The cholinergic neurons in the septohippocampal projection are implicated in hippocampal functions such as spatial learning and memory. The aim of this study was to examine how septohippocampal cholinergic transmission is modulated by muscarinic inputs and by the neuropeptide galanin, co-localized with acetylcholine (ACh) in septohippocampal cholinergic neurons, and how spatial learning assessed by the Morris water maze test is affected. Muscarinic inputs to the septal area are assumed to be excitatory, whereas galanin is hypothesized to inhibit septohippocampal cholinergic function. To test these hypotheses, compounds were microinjected into the medial septum and hippocampal ACh release was assessed by microdialysis probes in the ventral hippocampus of the rat. Blockade of septal muscarinic transmission by intraseptal scopolamine increased hippocampal ACh release suggesting that septal cholinergic neurons are under tonic inhibition. Stimulation of septal muscarinic receptors by carbachol also increased hippocampal ACh release. Despite this increase, both scopolamine and carbachol tended to impair hippocampus-dependent spatial learning. This finding also suggests a revision of the simplistic notion that an increase in hippocampal ACh may be facilitatory for learning and memory. Galanin infused into the medial septum enhanced hippocampal ACh release and facilitated spatial learning, suggesting that septal galanin, contrary to earlier claims, does not inhibit but excites septohippocampal cholinergic neurons. Galanin receptor stimulation combined with muscarinic blockade in the septal area resulted in an excessive increase of hippocampal ACh release combined with an impairment of spatial learning. This finding suggests that the level of muscarinic activity within the septal area may determine the effects of galanin on hippocampal cognitive functions. In summary, a limited range of cholinergic muscarinic transmission may contribute to optimal hippocampal function, a finding that has important implications for therapeutic approaches in the treatment of disorders of memory function.

Hippocampal dynorphin B injections impair spatial learning in rats: a κ-opioid receptor-mediated effect

The hippocampus plays a central role in the acquisition and storage of information. Long-term potentiation in the mossy fibre pathway to the CA3 region in the hippocampus, an animal model of memory acquisition, is modulated by dynorphin peptides. This study investigated the possible role of hippocampal dynorphin in spatial learning. Male rats were trained in the Morris Water Task after microinjection with different doses of dynorphin B (1, 3.3 or 10 nmol/rat) or artificial cerebrospinal fluid (as control) into the CA3 region of the dorsal hippocampus. Dynorphin B was found to impair spatial learning at all tested doses. The synthetic kappa1-selective opiate receptor antagonist nor-binaltorphimine (2 nmol) also given into the hippocampus fully blocked the acquisition impairment caused by dynorphin B (10 nmol), while nor-binaltorphimine alone did not affect learning performance. These findings suggest that dynorphin peptides could play a modulatory role in hippocampal plasticity by acting on hippocampal kappa-receptors and thereby impair spatial learning.

Nociceptin/orphanin FQ metabolism and bioactive metabolites

The endogenous ligand for the orphan NOR receptor (earlier named ORL1) was recently discovered. This ligand, nociceptin/orphanin FQ is involved in a number of pharmacological actions in the CNS, including modulation of pain and cognition. However, its specific physiological role remains to be determined. Two major pathways of metabolism have been identified; the action of aminopeptidase(s) that prominently occurs in plasma, and endopeptidase activity that successively generates the N-terminal 1-13 and 1-9 fragments. Both pathways result in fragments that are inactive at the NOR receptor. However, short N-terminal fragments appear to be active in blocking the release of substance P from primary afferent C-fiber terminals in the dorsal spinal cord. The same endopeptidase(s) may also be involved in the fragmentation of dynorphin A since the inhibitor profile is similar. Enzyme activity is upregulated by morphine using either peptide as substrate that may lead to pharmacological interactions.

Nociceptin/orphanin FQ modulates spatial learning via ORL-1 receptors in the dorsal hippocampus of the rat.

The endogenous peptide nociceptin (orphanin FQ) plays a role in several important physiological functions in the CNS such as pain, anxiety and locomotion. It has previously been found that injection of 10 nmol nociceptin into the CA3 region of the hippocampus markedly impairs spatial learning and memory in the rat. The present study examined the effects of lower doses of nociceptin (3.3, 1, 0.33 and 0.1 nmol/rat) on spatial learning. The 3.3 nmol dose impaired spatial learning over the 5 days of training although the effect was not as strong as with 10 nmol. In contrast, the two lower doses, 1 and 0.33 nmol/rat, improved spatial learning whereas the lowest dose, 0.1 nmol/rat, had no significant effect. Both the impairing and facilitating effect of nociceptin could be blocked by an ORL-1 receptor antagonist, [Phe1Psi(CH(2)-NH)Gly2]NC(1-13)NH2 (10 nmol/rat), indicating that both effects are ORL-1 receptor-mediated. The 3.3 nmol dose of nociceptin did not impair the performance in the visual platform task and did not alter swim speed or motor activity indicating no effects on motivation or motor performance. Taken together, these results show that nociceptin has a biphasic dose-effect curve and provide further evidence for a role of this neuropeptide in cognitive processes in the hippocampus.

Evidence in locomotion test for the functional heterogeneity of ORL-1 receptors

The ORL1 agonists nociceptin and Ro 64‐6198 were compared in their ability to modify spontaneous locomotor activity in male NMRI mice not habituated to the test environment. Higher doses of nociceptin (>5 nmol i.c.v.) reduced whereas lower doses (<1 nmol i.c.v.) stimulated locomotor activity. Both effects were blocked by the putative ORL1 antagonists [NPhe1]nociceptin(1–13)NH2 (10 nmol i.c.v.) and UFP101 (10 nmol, i.c.v.). The effects were also blocked by naloxone benzoylhydrazone (1 mg kg−1 s.c.), but not by the nonselective opioid antagonist naloxone (1 mg kg−1 s.c.). In contrast to nociceptin, the synthetic ORL1 agonist Ro 64‐6198 (0.01–1.0 mg kg−1 i.p.) produced monophasic inhibition of locomotor activity, which was insensitive to the treatment with [NPhe1]nociceptin(1–13)NH2 or naloxone benzoylhydrazone. Treatment with UFP101 abolished the locomotor inhibition induced by Ro 64‐6198 (1.0 mg kg−1), whereas naloxone (1.0 mg kg−1, s.c.) further increased the locomotor‐inhibitory effects. Naloxone benzoylhydrazone (0.3; 1.0 and 3.0 mg kg−1 s.c.) increased locomotor activity, although the effect was statistically significant only with the highest dose used. Pretreatment with the tyrosine hydroxylase inhibitor H44‐68 totally eliminated the motor‐stimulatory effects of low doses of nociceptin, probably via dopamine depletion. The results suggest that nociceptin stimulates locomotor activity at low doses if dopamine activity is intact. High doses of nociceptin and all the tested doses of Ro 64‐6198 seem to interact with a functionally different subset of ORL1 receptors. In addition, the effects of Ro 64‐6198 are modulated by tonic opioid receptor activity.

Evidence that N-terminal fragments of nociceptin modulate nociceptin-induced scratching, biting and licking in mice

The intrathecal (i.t.) injection of 3.0 fmol nociceptin (orphanin FQ) elicited scratching, biting and licking responses in mice. N-terminal fragments of nociceptin, nociceptin (1-7), nociceptin (1-9) and nociceptin (1-13), induced no characteristic behavioral response. When these N-terminal fragments of nociceptin were injected simultaneously with nociceptin, the behavioral response induced by nociceptin was reduced dose-dependently. Nociceptin (1-13) was much more potent than nociceptin (1-7) and nociceptin (1-9) and antagonized nociceptin-induced response at equimolar doses. No significant effects of the N-terminal fragments were observed against the scratching, biting and licking response elicited by i.t. administration of substance P or N-methyl-D-aspartate. These results suggest that N-terminal fragments formed endogenously in the spinal cord may have an antagonistic effect on nociceptin-induced behavioral responses.

Profound but transient deficits in learning and memory after global ischemia using a novel water maze test

The pyramidal CA1 neurons of the hippocampus are critically involved in spatial learning and memory. These neurons are especially vulnerable to cerebral ischemia, but in spite of this, it has been consistently difficult to show any learning and memory deficits in two-vessel occlusion models of global ischemia. Transient global ischemia was induced in adult male rats under general anaesthesia administered by artificial respiration to prevent respiratory arrest. Systemic blood pressure was reduced to below 50 mmHg by instant adjustments of the halothane concentration, before and during bilateral occlusion of the carotid arteries. Cerebral blood flow was monitored by laser-Doppler flowmetry. Dying neurons were detected by TUNEL at 14 days after ischemia and surviving neurons by NeuN at 14 and 125 days after ischemia. Learning and memory was assessed in a novel water maze with three successive left-right choices. Transient global ischemia produced a profound and selective degeneration of CA1 neurons at 14 days after ischemia. This degeneration was associated with severe impairments in learning at 13 days after ischemia and in memory, as tested 24 h afterwards. At 125 days after ischemia, there was no significant learning and memory impairment, whereas the number of CA1 neurons was increased. These results show that transient global ischemia induced by two-vessel occlusion may lead to severe, but transient, impairments in learning and memory using a novel water maze, and that restored learning and memory is associated with an increased number of CA1 neurons.

In vivo metabolism of nociceptin/orphanin FQ in rat hippocampus.

The in vivo metabolism of the newly identified endogenous ligand for the ORL1 receptor, the opioid-like peptide nociceptin (orphanin FQ) in rat hippocampus was studied using size-exclusion chromatography linked to electrospray ionization mass spectrometry. The results show that nociceptin is metabolized step-wise in vivo into fragments (1-13) and (14-17) as well as (1-9) and (10-13), respectively. Interestingly, the (1-13) and (1-9) fragments have the same C-terminus, Arg-Ala-Lys, suggesting that this is a motif recognized by an enzyme which fragments the peptide in two consecutive steps. Injection of the (1-13) fragment into rat hippocampus had no effect on spatial learning or motor function under conditions where nociceptin is active, showing that this metabolic conversion reduces affinity for the ORL-1-receptor.