J. Del Río

Effects of maternal separation on hypothalamic–pituitary–adrenal responses, cognition and vulnerability to stress in adult female rats

We studied the long term effects of neonatal stress in female rats and subsequent responses to stress when adults. Female rats that experienced maternal separation (MS) showed in adulthood depressive-like behavior in the forced swimming test and cognitive impairments in the novel object recognition test, which were reverted by the glucocorticoid receptor antagonist mifepristone or the beta-adrenoceptor antagonist propranolol. Markers of HPA axis (corticosterone levels, CRF mRNA levels in the paraventricular nucleus and glucocorticoid receptor density in the hippocampus) were altered by MS, suggesting that an altered HPA axis function may be associated to behavioral and cognitive deficits in MS female rats. In addition, MS rats were found to be more vulnerable to chronic stress than controls as shown by decreases in open field activity, increases in immobility time in the forced swim test, and changes in markers of HPA axis (decreases in the density of glucocorticoid receptors). These present findings are discussed in terms of gender differences in adulthood.

Enhanced anxiety, depressive-like behaviour and impaired recognition memory in mice with reduced expression of the vesicular glutamate transporter 1 (VGLUT1)

Three isoforms of a vesicular glutamate transporter (VGLUT1–3) have been identified. Of these, VGLUT1 is the major isoform in the cerebral cortex and hippocampus where it is selectively located on synaptic vesicles of excitatory glutamatergic terminals. Variations in VGLUT1 expression levels have a major impact on the efficacy of glutamate synaptic transmission. Given evidence linking alterations in glutamate neurotransmission to various neuropsychiatric disorders, we investigated the possible influence of a down‐regulation of VGLUT1 transporter on anxiety, depressive‐like behaviour and learning. The behavioural phenotype of VGLUT1‐heterozygous mice (C57BL/6) was compared to wild‐type (WT) littermates. Moreover, VGLUT1–3 expression, hippocampal excitatory terminal ultrastructure and neurochemical phenotype were analysed. VGLUT1‐heterozygous mice displayed normal spontaneous locomotor activity, increased anxiety in the light–dark exploration test and depressive‐like behaviour in the forced swimming test: no differences were shown in the elevated plus‐maze model of anxiety. In the novel object recognition test, VGLUT1+/– mice showed normal short‐term but impaired long‐term memory. Spatial memory in the Morris water maze was unaffected. Western blot analysis confirmed that VGLUT1 heterozygotes expressed half the amount of transporter compared to WT. In addition, a reduction in the reserve pool of synaptic vesicles of hippocampal excitatory terminals and a 35–45% reduction in GABA in the frontal cortex and the hippocampus were observed in the mutant mice. These observations suggest that a VGLUT1‐mediated presynaptic alteration of the glutamatergic synapses, in specific brain regions, leads to a behavioural phenotype resembling certain aspects of psychiatric and cognitive disorders.

Extensive loss of brain dopamine and serotonin induced by chronic administration of MPTP in the marmoset

Common marmosets were given a subcutaneous injection of MPTP (1.25-2.5 mg/kg twice a week) for 5 or 10 consecutive months and were sacrificed after a survival time of 6 months or 15 days, respectively. The parkinsonian symptoms were not very marked at the time of sacrifice but there was a strong decrease of dopamine and, to a lesser extent, of its metabolites in the striatum and in some extrastriatal regions. There was also a profound loss of serotonin in the striatum and in all of the extrastriatal regions analyzed, which was still highly significant 6 months after discontinuation of MPTP treatment. The results suggest that the selected dosage schedule produces a widespread and lasting neuronal degeneration closely resembling the neurochemical pathology of Parkinsons disease.

Involvement of GABA systems in acetylcholine release induced by 5-HT3 receptor blockade in slices from rat entorhinal cortex.

The aim of the present study was to examine the role of 5-HT3 receptors in spontaneous and K(+)-evoked acetylcholine (ACh) release from rat entorhinal cortex and striatal slices. The 5-HT3 receptor antagonists ondansetron and granisetron (0.01-10 microM) produced a concentration-dependent increase in both spontaneous and K(+)-evoked [3H]ACh release in the two brain regions studied. The release of ACh was Ca(2+)-dependent and tetrodotoxin-sensitive. 5-HT3 receptor agonists, such as 2-methyl-5-HT and 1-phenylbiguanide, at concentrations up to 1 microM, did not show any intrinsic effect on [3H]ACh release in both rat brain regions. However, 2-methyl-5-HT, 1 microM, fully blocked the ondansetron-induced enhancement in both basal and K(+)-evoked ACh release, suggesting that 5-HT, through 5-HT3 receptor activation, tonically inhibits ACh release. The possible implication of interposed inhibitory systems in ACh release after 5-HT3 receptor blockade was subsequently analyzed. While the effect of ondansetron was not modified by haloperidol or naloxone, the GABAA receptor antagonist bicuculline produced a marked potentiation of ACh release in the entorhinal cortex but not in the striatum. The results suggest that in this cortical area 5-HT activates 5-HT3 receptors located on GABAergic neurons which in turn inhibit cholinergic function.

Anxiogenic-like effects and reduced stereological counting of immunolabelled 5-hydroxytryptamine6 receptors in rat nucleus accumbens by antisense oligonucleotides

The physiological role of 5-hydroxytryptamine6 receptors in the central nervous system has not yet been elucidated. The high affinity of various psychotropic drugs for 5-hydroxytryptamine6 receptors has led to the suggestion that this receptor type may be a novel target in neuropsychiatry. We have found that continuous intracerebroventricular administration of a 5-hydroxytryptamine6 receptor antisense oligonucleotide, but not of a missense oligonucleotide, produced an anxiogenic-like response in rats using two different models of anxiety, the social interaction test and the elevated plus-maze. Neither oligonucleotide treatment modified locomotor activity, rectal temperature or food intake, suggesting a low or null neurotoxicity. The effectiveness of the treatment with the designed antisense oligonucleotide to block the synthesis of the protein encoded by the target mRNA was assessed by immunolabelling 5-hydroxytryptamine6 receptors in the nucleus accumbens, where this receptor is highly expressed, using previously characterized specific antibodies. The density of the immunostaining was quantified by means of an unbiased three-dimensional stereologic procedure, which revealed a significant reduction (-25%) in the number of immunolabelled neuronal elements. These results suggest that, in addition to other 5-hydroxytryptamine receptor subtypes, 5-hydroxytryptamine6 receptors in the nucleus accumbens may participate in anxiety-related neurobiological mechanisms.

Involvement of neurokinins in the non-cholinergic response to activation of 5-HT3 and 5-HT4 receptors in guinea-pig ileum

1 The involvement of neurokinins in the non‐cholinergically‐mediated contractile response induced by stimulation of 5‐HT3 and 5‐HT4 receptors has been examined in the longitudinal muscle‐myenteric plexus preparation of the guinea‐pig ileum. 2 The 5‐HT3 receptor agonist, 2‐methyl‐5‐hydroxytryptamine (2‐methyl‐5‐HT), showed a lower potency in this preparation than the more selective 5‐HT4 receptor agonist 5‐methoxytryptamine. The effect of both drugs was markedly reduced by atropine. 3 Substance P (SP) and neurokinin B (NKB) produced biphasic concentration‐response curves in the preparation. Neurokinin A (NKA), the NK1 receptor agonist, [Sar9, Met(O2)11]SP and the NK3 receptor agonist, senktide yielded monophasic concentration‐response curves. 4 After desensitization of the NK1 receptor with SP or [Sar9, met(O2)11]SP, in the presence of atropine, the contractile response to 2‐methyl‐5‐HT was entirely blocked. Desensitization of NK3 receptors with NKB, also in the presence of atropine, fully suppressed the 5‐HT4 receptor‐mediated contraction evoked by 5‐methoxytryptamine. 5 In preparations prelabelled with [3H]‐choline, SP produced a concentration‐dependent increase in tritium overflow, an index of [3H]‐acetylcholine release, while an inverse relationship was found with NKB. At low neurokinin concentrations, the releasing effect of NKB was much more marked. 6 It is suggested that in the response to 5‐HT3 receptor stimulation, there is a role for SP and acetylcholine. NKB appears to be preferentially involved in the release of acetylcholine elicited by stimulation of 5‐HT4 receptors.

GM-1 ganglioside promotes the recovery of surviving midbrain dopaminergic neurons in MPTP-treated monkeys.

We have examined the influence of chronic GM-1 treatment (20 mg/kg i.m. for 16 consecutive days) on the extent of dopaminergic damage induced by acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration in cynomolgus monkeys using immunohistochemical and neurochemical analysis. The total number of tyrosine hydroxylase-immunoreactive neurons was reduced in different catecholaminergic mesencephalic regions of MPTP-treated monkeys such as substantia nigra pars compacta, mainly in the ventral portion of the nucleus (39% reduction), substantia nigra pars lateralis (31%), peri- and retrorubral catecholaminergic cell group and ventral tegmental area (A8 and A10 respectively, 20% reduction). A similar degree of neuronal loss was observed in the MPTP+GM-1-treated animals, suggesting that GM-1 ganglioside does not exert a protective effect against MPTP-induced dopaminergic cell loss. Moreover, no neurochemical recovery from the striatal dopaminergic depletion induced by MPTP was found after GM-1 treatment. However, the optical density of tyrosine hydroxylase fibers and the cellular tyrosine hydroxylase content were increased in the substantia nigra pars compacta and ventral tegmental area of the MPTP-treated monkeys which received GM-1 ganglioside, compared with animals treated only with the neurotoxin. These results indicate that GM-1 does not protect against cell death but exerts a neurotrophic effect on surviving dopaminergic neurons in the midbrain of MPTP-lesioned monkeys, suggesting that GM-1 ganglioside may be potentially useful for the treatment of neurodegenerative disorders such as Parkinsons disease.

MPTP-induced parkinsonism in primates : pattern of striatal dopamine loss following acute and chronic administration

We analyzed the effect of two different schedules of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment on dopaminergic systems in the striatum of cynomolgus monkeys. Acute MPTP treatment produced a marked dopamine (DA) depletion, more severe in the caudate nucleus than in the putamen. Chronic MPTP induced a more pronounced reduction in DA levels, the putamen being slightly more affected than the caudate nucleus, in accord with immunohistochemical findings that showed a higher loss of tyrosine-hydroxylase positive neurons in ventral subpopulations of the substantia nigra pars compacta. A striking increment in the quotient DOPAC+HVA/DA was also observed in chronically but not in acutely treated monkeys, especially in the putamen. In chronically treated animals there was a nearly complete loss of DA in all subdivisions of the putamen. In the caudate nucleus, a rostrocaudal gradient of DA depletion was found, with a greater decrease in DA concentration in the rostral parts, especially in the dorsolateral portions. The pattern of striatal DA loss characteristic of Parkinsons disease can be reproduced to a certain extent in MPTP-intoxicated primates.

Opposing effects of AMPA and 5-HT1A receptor blockade on passive avoidance and object recognition performance: Correlation with AMPA receptor subunit expression in rat hippocampus

It has been suggested that antagonists at serotonin 5-HT1A receptors may exert a procognitive effect by facilitating glutamatergic neurotransmission. Here we further explored this issue by looking for the ability of a 5-HT1A antagonist to prevent the learning deficit induced by AMPA receptor blockade in two behavioural procedures in rats, and for concomitant molecular changes presumably involved in memory formation in the hippocampus. Pretraining administration of the competitive AMPA receptor antagonist, NBQX, produced a dose-related retention impairment in a passive avoidance task 24h later, and also impaired retention in a novel object recognition test when an intertrial interval of 3h was selected. Pretreatment with the selective 5-HT1A receptor antagonist, WAY-100635, prevented the learning deficit induced by NBQX in the two behavioural procedures. In biochemical studies performed on rat hippocampus after the retention tests, we found that learning increased the membrane levels of AMPA receptor GluR1 and GluR2/3 subunits, as well as the phosphorylated forms of GluR1, effects that were abolished by NBQX administration before the training session. Pretreatment with WAY-100635 counteracted the NBQX effects and restored the initial learning-specific increase in Ca2+/calmodulin-dependent protein kinase II (CaMKII) function and the later increase in GluR2/3 and phosphorylated GluR1 surface expression. Moreover, administration of WAY-100635 before object recognition training improved recognition memory 24h later and potentiated the learning-associated increase in AMPA receptor subunits. The results support the proposed utility of 5-HT1A antagonists in the treatment of cognitive disorders.

The pharmacology of VA21B7: an atypical 5-HT3 receptor antagonist with anxiolytic-like properties in animal models

VA21B7 (3-[2-(4′-piperonylpiperazinyl) indolyl] carboxaldehyde) was synthesized as a potential 5-HT3 receptor antagonist. Even though VA21B7 showed a higher affinity towards 5-HT3 receptors as compared to other receptors studied, it was not a potent 5-HT3 receptor antagonist either in the periphery or in the brain. In a simple animal model of anxiety such as the two-compartment box in mice, a remarkable anxiolytic-like effect was found at doses of 2–500 µg/kg IP and also at low oral doses, in the microgram range. These drug doses did not produce any significant effect on spontaneous motor activity of mice. The anxiolytic profile of VA21B7 was further explored using other models of anxiety in rats such as the elevated plus-maze and punished-drinking. VA21B7 was compared with standard 5-HT3 receptor antagonists such as ondansetron, tropisetron and granisetron, with the 5-HT1A agent buspirone and with diazepam. In the plus-maze, VA21B7 showed an anxiolytic-like profile after doses of 0.25–0.5 mg/kg IP or 2–4 mg/kg PO which did not modify the number of total entries into the open and closed arms of the maze. Diazepam, granisetron and tropisetron were also effective in this test but not ondansetron and buspirone. VA21B7 was also able to release suppressed behaviour in the punished-drinking test. The dose-response curve was bell-shaped with a peak at 2–4 mg/kg. At variance with other studies, 5-HT3 receptor antagonists also increased the number of shocks taken in this test and the dose-response curve was also bell-shaped. VA21B7 was not anticonvulsant like diazepam, its anxiolytic action in the light/dark test was not flumazenil-sensitive and there was no rebound anxiogenic effect on withdrawal from chronic VA21B7 treatment for 15 consecutive days. Moreover, VA21B7 was not amnesic like the benzodiazepines but low doses of 2–4 mg/kg reduced the memory deficits induced in rats by scopolamine. Much higher doses were necessary to decrease spontaneous motor activity in rats. Since VA21B7 appears to be well tolerated in rodents at high doses, we think that it is of potential interest as an anxiolytic in humans.