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Dehydroepiandrosterone (DHEA) and its metabolites, DHEA-sulphate (DHEA-S) and androsterone, have neurosteroid activity. In this study, we examined whether DHEA, DHEA-S and androsterone, can influence serotonin (5-HT) neuronal firing activity via modulation of γ-aminobutryic acid (GABAA) receptors. The firing of presumed 5-HT neurones in a slice preparation containing rat dorsal raphe nucleus was inhibited by the GABAA receptor agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridinyl-3-ol (THIP) (25 μM) and GABA (100 μM). DHEA (100 and 300 μM) and DHEA-S (1, 10 and 100 μM) caused a rapid and reversible attenuation of the response to THIP. DHEA (100 μM) and DHEA-S (100 μM) also attenuated the effect of GABA. Androsterone (10 and 30 μM) markedly enhanced the inhibitory response to THIP (25 μM). The effect was apparent during androsterone administration but persisted and even increased in magnitude after drug wash-out. The data indicate that GABAA receptor-mediated regulation of 5-HT neuronal firing is sensitive to negative modulation by DHEA and its metabolite DHEA-S is sensitive to positive modulation by the metabolite androsterone. The effects of these neurosteroids on GABA A receptor-mediated regulation of 5-HT firing may underlie some of the reported behavioural and psychological effects of endogenous and exogenous DHEA. [ABSTRACT FROM PUBLISHER]

We recently reported that the central nucleus of the inferior colliculus (the auditory midbrain) is innervated by glutamatergic pyramidal cells originating not only in auditory cortex (AC), but also in multiple 'non-auditory' regions of the cerebral cortex. Here, in anaesthetised rats, we used optogenetics and electrical stimulation, combined with recording in the inferior colliculus to determine the functional influence of these descending connections. Specifically, we determined the extent of monosynaptic excitation and the influence of these descending connections on spontaneous activity in the inferior colliculus. A retrograde virus encoding both green fluorescent protein (GFP) and channelrhodopsin (ChR2) injected into the central nucleus of the inferior colliculus (ICc) resulted in GFP expression in discrete groups of cells in multiple areas of the cerebral cortex. Light stimulation of AC and primary motor cortex (M1) caused local activation of cortical neurones and increased the firing rate of neurones in ICc indicating a direct excitatory input from AC and M1 to ICc with a restricted distribution. In naïve animals, electrical stimulation at multiple different sites within M1, secondary motor, somatosensory, and prefrontal cortices increased firing rate in ICc. However, it was notable that stimulation at some adjacent sites failed to influence firing at the recording site in ICc. Responses in ICc comprised singular spikes of constant shape and size which occurred with a short, and fixed latency (∼ 5 ms) consistent with monosynaptic excitation of individual ICc units. Increasing the stimulus current decreased the latency of these spikes, suggesting more rapid depolarization of cortical neurones, and increased the number of (usually adjacent) channels on which a monosynaptic spike was seen, suggesting recruitment of increasing numbers of cortical neurons. Electrical stimulation of cortical regions also evoked longer latency, longer duration increases in firing activity, comprising multiple units with spikes occurring with significant temporal jitter, consistent with polysynaptic excitation. Increasing the stimulus current increased the number of spikes in these polysynaptic responses and increased the number of channels on which the responses were observed, although the magnitude of the responses always diminished away from the most activated channels. Together our findings indicate descending connections from motor, somatosensory and executive cortical regions directly activate small numbers of ICc neurones and that this in turn leads to extensive polysynaptic activation of local circuits within the ICc., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier B.V.)

The anti-inflammatory drug salicylate induces tinnitus in animals and man. Salicylate reduces cochlear output but causes hyperactivity in higher auditory centres, including the inferior colliculus (the auditory midbrain). Using multi-electrode recording in anaesthetised guinea pigs (Cavia porcellus), we addressed the hypothesis that salicylate-induced hyperactivity in the inferior colliculus involves nitric oxide signalling secondary to increased ascending excitatory input. Systemic salicylate (200 mg/kg i.p., 0 h) markedly increased spontaneous and sound-driven neuronal firing in the inferior colliculus (3-6 h post drug), with both onset and sustained responses to pure tones being massively increased. Reverse microdialysis of increasing concentrations of salicylate directly into the inferior colliculus (100 µM-10 mM, from 0 h) failed to mimic systemic salicylate. In contrast, it caused a small, transient, increase in sound-driven firing (1 h), followed by a larger sustained decrease in both spontaneous and sound-driven firing (2-5 h). When salicylate was given systemically, reverse microdialysis of the neuronal nitric oxide synthase inhibitor L-methyl arginine into the inferior colliculus (500 mM, 2-6 h) completely blocked the salicylate-induced increase in spontaneous and sound-driven neuronal firing. Our data indicate that systemic salicylate induces neuronal hyperactivity in the auditory midbrain via a mechanism outside the inferior colliculus, presumably upstream in the auditory pathway; and that the mechanism is ultimately dependent on nitric oxide signalling within the inferior colliculus. Given that nitric oxide is known to mediate NMDA receptor signalling in the inferior colliculus, we propose that salicylate activates an ascending glutamatergic input to the inferior colliculus and that this is an important mechanism underlying salicylate-induced tinnitus., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022. Published by Elsevier B.V.)

The role of dopamine in regulating sleep-state transitions during, both natural sleep and under anaesthesia, is still unclear. Recording in vivo in the rat mPFC under urethane anaesthesia, we observed predominantly slow wave activity (SWA) of <1 Hz in the local field potential interrupted by occasional spontaneous transitions to a low-amplitude-fast (LAF) pattern of activity. During periods of SWA, transitions to LAF activity could be rapidly and consistently evoked by electrical stimulation of the ventral tegmental area (VTA). Spontaneous LAF activity, and that evoked by stimulation of the VTA, consisted of fast oscillations similar to those seen in the rapid eye movement (REM)-like sleep state. Spontaneous and VTA stimulation-evoked LAF activity occurred simultaneously along the dorsoventral extent of all mPFC subregions. Evoked LAF activity depended on VTA stimulation current and could be elicited using either regular (25-50 Hz) or burst stimulation patterns and was reproducible upon repeated stimulation. Simultaneous extracellular single-unit recordings showed that during SWA, presumed pyramidal cells fired phasically and almost exclusively on the Up state, while during both spontaneous and VTA-evoked LAF activity, they fired tonically. The transition to LAF activity evoked by VTA stimulation depended on dopamine D 1 -like receptor activation as it was almost completely blocked by systemic administration of the D 1 -like receptor antagonist SCH23390. Overall, our data demonstrate that activation of dopamine D 1 -like receptors in the mPFC is important for regulating sleep-like state transitions., (© 2020 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Neuronal nitric oxide synthase (nNOS) catalyses the production of the neurotransmitter nitric oxide. nNOS is expressed in the dorsal raphe nucleus (DRN), a source of ascending serotonergic projections. In this study, we examined the distribution nNOS and the function of nitric oxide in the DRN and adjacent median raphe nucleus (MRN) of the rat. We hypothesized that nNOS is differentially expressed across the raphe nuclei and that nitric oxide influences the firing activity of a subgroup of 5-HT neurons. Immunohistochemistry revealed that, nNOS is present in around 40% of 5-HT neurons, throughout the DRN and MRN, as well as in some non-5-HT neurons immediately adjacent to the DRN and MRN. The nitric oxide receptor, soluble guanylyl cyclase, was present in all 5-HT neurons examined in the DRN and MRN. In vitro extracellular electrophysiology revealed that application of the nitric oxide donor, diethylamine NONOate (30-300 µM) inhibited 60%-70% of putative 5-HT neurons, excited approximately 10% of putative 5-HT neurons and had no effect on the rest. The inhibitory response to nitric oxide was blocked by [1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ, 30 or 100 µM), indicating mediation by soluble guanylyl cyclase. Juxtacellular labelling revealed that nitric oxide inhibits firing in both putative 5-HT neurons which express nNOS and those which do not express nNOS. Our data are consistent with the notion that nitric oxide acts as both a trans-synaptic and autocrine signaller in 5-HT neurons in the DRN and MRN and that its effects are widespread and primarily inhibitory., (© 2020 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Our perceptual experience of sound depends on the integration of multiple sensory and cognitive domains, however the networks subserving this integration are unclear. Connections linking different cortical domains have been described, but we do not know the extent to which connections also exist between multiple cortical domains and subcortical structures. Retrograde tracing in adult male rats ( Rattus norvegicus ) revealed that the inferior colliculus, the auditory midbrain, receives dense descending projections not only, as previously established, from the auditory cortex, but also from the visual, somatosensory, motor, and prefrontal cortices. While all these descending connections are bilateral, those from sensory areas show a more pronounced ipsilateral dominance than those from motor and prefrontal cortices. Injections of anterograde tracers into the cortical areas identified by retrograde tracing confirmed those findings and revealed cortical fibers terminating in all three subdivisions of the inferior colliculus. Immunolabeling showed that cortical terminals target both GABAergic inhibitory, and putative glutamatergic excitatory neurons. These findings demonstrate that auditory perception and behavior are served by a network that includes extensive descending connections to the midbrain from sensory, behavioral, and executive cortices. SIGNIFICANCE STATEMENT Making sense of what we hear depends not only on the analysis of sound, but also on information from other senses together with the brain's predictions about the properties and significance of the sound. Previous work suggested that this interplay between the senses and the predictions from higher cognitive centers occurs within the cerebral cortex. By tracing neural connections in rat, we show that the inferior colliculus, the subcortical, midbrain center for hearing, receives extensive connections from areas of the cerebral cortex concerned with vision, touch, movement, and cognitive function, in addition to areas representing hearing. These findings demonstrate that wide-ranging cortical feedback operates at an earlier stage of the hearing pathway than previously recognized., (Copyright © 2019 the authors.)

Nitric oxide (NO) is a neurotransmitter synthesized in the brain by neuronal nitric oxide synthase (nNOS). Using immunohistochemistry and confocal imaging in the inferior colliculus (IC, auditory midbrain) of the guinea pig ( Cavia porcellus , male and female), we show that nNOS occurs in two distinct cellular distributions. We confirm that, in the cortices of the IC, a subset of neurons show cytoplasmic labeling for nNOS, whereas in the central nucleus (ICc), such neurons are not present. However, we demonstrate that all neurons in the ICc do in fact express nNOS in the form of discrete puncta found at the cell membrane. Our multi-labeling studies reveal that nNOS puncta form multiprotein complexes with NMDA receptors, soluble guanylyl cyclase (sGC), and PSD95. These complexes are found apposed to glutamatergic terminals, which is indicative of synaptic function. Interestingly, these glutamatergic terminals express both vesicular glutamate transporters 1 and 2 denoting a specific source of brainstem inputs. With in vivo electrophysiological recordings of multiunit activity in the ICc, we found that local application of NMDA enhances sound-driven activity in a concentration-dependent and reversible fashion. This response is abolished by blockade of nNOS or sGC, indicating that the NMDA effect is mediated solely via the NO and cGMP signaling pathway. This discovery of a ubiquitous, but highly localized, expression of nNOS throughout the ICc and demonstration of the dramatic influence of the NMDA activated NO pathway on sound-driven neuronal activity imply a key role for NO signaling in auditory processing. SIGNIFICANCE STATEMENT We show that neuronal nitric oxide synthase (nNOS), the enzyme that synthesizes nitric oxide (NO), occurs as puncta in apparently all neurons in the central nucleus of the inferior colliculus (ICc) in the auditory midbrain. Punctate nNOS appears at glutamatergic synapses in a complex with glutamate NMDA receptors (NMDA-Rs), soluble guanylyl cyclase (sGC, the NO receptor), and PSD95 (a protein that anchors receptors and enzymes at the postsynaptic density). We show that NMDA-R modulation of sound-driven activity in the ICc is solely mediated by activation of nNOS and sGC. The presence of nNOS throughout this sensory nucleus argues for a major role of NO in hearing. Furthermore, this punctate form of nNOS expression may exist and have gone unnoticed in other brain regions., (Copyright © 2019 the authors 0270-6474/19/390876-12$15.00/0.)

Cortical slow oscillations (0.1-1 Hz), which may play a role in memory consolidation, are a hallmark of non-rapid eye movement (NREM) sleep and also occur under anesthesia. During slow oscillations the neuronal network generates faster oscillations on the active Up-states and these nested oscillations are particularly prominent in the PFC. In rodents the medial prefrontal cortex (mPFC) consists of several subregions: anterior cingulate cortex (ACC), prelimbic (PrL), infralimbic (IL), and dorsal peduncular cortices (DP). Although each region has a distinct anatomy and function, it is not known whether slow or fast network oscillations differ between subregions in vivo. We have simultaneously recorded slow and fast network oscillations in all four subregions of the rodent mPFC under urethane anesthesia. Slow oscillations were synchronous between the mPFC subregions, and across the hemispheres, with no consistent amplitude difference between subregions. Delta (2-4 Hz) activity showed only small differences between subregions. However, oscillations in the spindle (6-15 Hz)-, beta (20-30 Hz), gamma (30-80 Hz)-, and high-gamma (80-150 Hz)-frequency bands were consistently larger in the dorsal regions (ACC and PrL) compared with ventral regions (IL and DP). In dorsal regions the peak power of spindle, beta, and gamma activity occurred early after onset of the Up-state. In the ventral regions, especially the DP, the oscillatory power in the spindle-, beta-, and gamma-frequency ranges peaked later in the Up-state. These results suggest variations in fast network oscillations within the mPFC that may reflect the different functions and connectivity of these subregions. NEW & NOTEWORTHY We demonstrate, in the urethane-anesthetized rat, that within the medial prefrontal cortex (mPFC) there are clear subregional differences in the fast network oscillations associated with the slow oscillation Up-state. These differences, particularly between the dorsal and ventral subregions of the mPFC, may reflect the different functions and connectivity of these subregions., (Copyright © 2017 the American Physiological Society.)

The neurotransmitter serotonin (5-HT) is involved in mood disorder aetiology and it has been reported that (organophosphate) OP exposure affects 5-HT turnover. The aim of this study was to elucidate the mechanism underlying OP effects on the adult 5-HT system. First, acute in vivo administration of the OP diazinon (0, 1.3, 13 or 39 mg/kg i.p.) to male Hooded Lister rats inhibited the activity of the cholinergic enzyme acetylcholinesterase in blood and in the hippocampus, dorsal raphe nucleus (DRN), striatum and prefrontal cortex. Diazinon-induced cholinesterase inhibition was greatest in the DRN, the brain's major source of 5-HT neurones. Second, acute in vivo diazinon exposure (0 or 39 mg/kg i.p.) increased the basal firing rate of DRN neurones measured ex vivo in brain slices. The excitatory responses of DRN neurones to α1-adrenoceptor or AMPA/kainate receptor activation were not affected by in vivo diazinon exposure but the inhibitory response to 5-HT was attenuated, indicating 5-HT1A autoreceptor down-regulation. Finally, direct application of the diazinon metabolite diazinon oxon to naive rat brain slices increased the firing rate of DRN 5-HT neurones, as did chlorpyrifos-oxon, indicating the effect was not unique to diazinon. The oxon-induced augmentation of firing was blocked by the nicotinic acetylcholine receptor antagonist mecamylamine and the AMPA/kainate glutamate receptor antagonist DNQX. Together these data indicate that 1) acute OP exposure inhibits DRN cholinesterase, leading to acetylcholine accumulation, 2) the acetylcholine activates nicotinic receptors on 5-HT neurones and also on glutamatergic neurones, thus releasing glutamate and activating 5-HT neuronal AMPA/kainate receptors 3) the increase in 5-HT neuronal activity, and resulting 5-HT release, may lead to 5-HT1A autoreceptor down-regulation. This mechanism may be involved in the reported increase in risk of developing anxiety and depression following occupational OP exposure., (Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Background: Patients with advanced Parkinson's disease (PD) often present with axial symptoms, including postural- and gait difficulties that respond poorly to dopaminergic agents. Although deep brain stimulation (DBS) of a highly heterogeneous brain structure, the pedunculopontine nucleus (PPN), improves such symptoms, the underlying neuronal substrate responsible for the clinical benefits remains largely unknown, thus hampering optimization of DBS interventions. Choline acetyltransferase (ChAT)::Cre(+) transgenic rats were sham-lesioned or rendered parkinsonian through intranigral, unihemispheric stereotaxic administration of the ubiquitin-proteasomal system inhibitor, lactacystin, combined with designer receptors exclusively activated by designer drugs (DREADD), to activate the cholinergic neurons of the nucleus tegmenti pedunculopontine (PPTg), the rat equivalent of the human PPN. We have previously shown that the lactacystin rat model accurately reflects aspects of PD, including a partial loss of PPTg cholinergic neurons, similar to what is seen in the post-mortem brains of advanced PD patients., Results: In this manuscript, we show that transient activation of the remaining PPTg cholinergic neurons in the lactacystin rat model of PD, via peripheral administration of the cognate DREADD ligand, clozapine-N-oxide (CNO), dramatically improved motor symptoms, as was assessed by behavioral tests that measured postural instability, gait, sensorimotor integration, forelimb akinesia and general motor activity. In vivo electrophysiological recordings revealed increased spiking activity of PPTg putative cholinergic neurons during CNO-induced activation. c-Fos expression in DREADD overexpressed ChAT-immunopositive (ChAT+) neurons of the PPTg was also increased by CNO administration, consistent with upregulated neuronal activation in this defined neuronal population., Conclusions: Overall, these findings provide evidence that functional modulation of PPN cholinergic neurons alleviates parkinsonian motor symptoms.

Occupational exposure to organophosphate (OPs) pesticides is reported to increase in the risk of developing anxiety and depression. Preclinical studies using OP levels, which inhibit acetylcholinesterase activity, support the clinical observations, but little is known of the effects of exposure below this threshold. We examined the effects of low level OP exposure on behaviours and neurochemistry associated with affective disorders. Adult rats were administered either diazinon (1 mg/kg i.p.) which is present in sheep dip and flea collars, chlorpyrifos (1 mg/kg i.p.) which is present in crop sprays, or vehicle for 5 days. OP exposure did not affect acetylcholinesterase activity (blood, cerebellum, caudate putamen, hippocampus, prefrontal cortex), anhedonia-like behaviour (sucrose preference), working memory (novel object recognition), locomotor activity or anxiety-like behaviour in the open field arena. In contrast OP exposure attenuated marble burying behaviour, an ethological measure of anxiety. The diazinon-induced reduction in marble burying persisted after exposure cessation. In comparison to vehicle, dopamine levels were lowered by chlorpyrifos, but not diazinon. 5-HT levels and turnover were unaffected by OP exposure. However, 5-HT transporter expression was reduced by diazinon suggesting subtle changes in 5-HT transmission. These data indicate exposure to occupational and domestic OPs, below the threshold to inhibit acetylcholinesterase, can subtly alter behaviour and neurochemistry., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Both the glutamatergic and serotonergic (5-HT) systems are implicated in the modulation of mood and anxiety. Descending cortical glutamatergic neurons regulate 5-HT neuronal activity in the midbrain raphe nuclei through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors. To analyze the functional role of GLUA1-containing AMPA receptors in serotonergic neurons, we used the Cre-ERT2/loxP-system for the conditional inactivation of the GLUA1-encoding Gria1 gene selectively in 5-HT neurons of adult mice. These Gria1(5-HT-/-) mice exhibited a distinct anxiety phenotype but showed no alterations in locomotion, depression-like behavior, or learning and memory. Increased anxiety-related behavior was associated with significant decreases in tryptophan hydroxylase 2 (TPH2) expression and activity, and subsequent reductions in tissue levels of 5-HT, its metabolite 5-hydroxyindoleacetic acid (5-HIAA), and norepinephrine in the raphe nuclei. However, TPH2 expression and activity as well as monoamine levels were unchanged in the projection areas of 5-HT neurons. Extracellular electrophysiological recordings of 5-HT neurons revealed that, while α1-adrenoceptor-mediated excitation was unchanged, excitatory responses to AMPA were enhanced and the 5-HT1A autoreceptor-mediated inhibitory response to 5-HT was attenuated in Gria1(5-HT-/-) mice. Our data show that a loss of GLUA1 protein in 5-HT neurons enhances AMPA receptor function and leads to multiple local molecular and neurochemical changes in the raphe nuclei that dysregulate 5-HT neuronal activity and induce anxiety-like behavior.

The attentional set shifting task (ASST) can be used to assess aspects of executive function, including reversal learning and set shifting. It has frequently been employed in between-subjects experimental designs: however, the clear advantages of within-subjects designs (reduction in variance, animal numbers, and cost) mean that investigation of the suitability of the ASST for within subjects designs is warranted. Rats were tested three times: test 1 (24 h after training), test 2 (24 h later), and test 3 (seven days later). On all three test days, animals showed the expected pattern of performance across the seven task stages with evidence of normal reversal learning (significant increase in trials to criterion when the rule was reversed) and intact set formation (significantly more trials to criterion for an extradimensional shift than for an intradimensional shift). There was a small decrease in total trials required to complete the task between test 1 and test 3 but this was not specific to any stage of the task. Latency to dig decreased on repeated testing suggesting some facilitation of associative learning. In conclusion, the rodent ASST is suitable for within-subject design longitudinal studies, increasing the utility and the translational value of this test and reducing numbers of animals needed in studies., (© The Author(s) 2014.)

Schizophrenia and bipolar disorder are associated with neurocognitive symptoms including deficits in attentional set shifting (changing attentional focus from one perceptual dimension to another) and reversal learning (learning a reversed stimulus/outcome contingency). Maternal infection during gestation and chronically flattened glucocorticoid rhythm are aetiological risk factors for schizophrenia and bipolar disorder. We hypothesised that these factors are causative in the neurocognitive deficits observed in schizophrenia and bipolar disorder. Here we used maternal immune activation (MIA) as a rat model of maternal infection, and sub-chronic low dose corticosterone treatment as a rat model of flattened glucocorticoid rhythm. For comparison we examined the effects of sub-chronic phencyclidine - a widely used rodent model of schizophrenia pathology. The effects of these three treatments on neurocognition were explored using the attentional set shifting task - a multistage test of executive functions. As expected, phencyclidine treatment selectively impaired set shifting ability. In contrast, MIA caused a marked and selective impairment of reversal learning. Corticosterone treatment impaired reversal learning but in addition also impaired rule abstraction and prevented the animals from forming an attentional set. The reversal learning deficits induced by MIA and corticosterone treatment were due to increases in non-perseverative rather than perseverative errors. Our data indicate that the cognitive deficits of schizophrenia and bipolar disorder may be explained by aetiological factors including maternal infection and glucocorticoid abnormalities and moreover suggest that the particular spectrum of cognitive deficits in individual patients may depend on the specific underlying aetiology of the disorder., (Copyright © 2013 Elsevier B.V. and ECNP. All rights reserved.)

Medial prefrontal cortex (mPFC) and orbitofrontal cortex (OFC) play critical roles in cognition and behavioural control. Glutamatergic, GABAergic, and monoaminergic dysfunction in the prefrontal cortex has been hypothesised to underlie symptoms in neuropsychiatric disorders. Here we characterised electrically-evoked field potentials in the mPFC and OFC. Electrical stimulation evoked field potentials in layer V/VI of the mPFC and layer V of the OFC. The earliest component (approximately 2 ms latency) was insensitive to glutamate receptor blockade and was presumed to be presynaptic. Later components were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX (20 µM)) and were assumed to reflect monosynaptic (latency 4-6 ms) and polysynaptic activity (latency 6-40 ms) mediated by glutamate via AMPA/kainate receptor. In the mPFC, but not the OFC, the monosynaptic component was also partly blocked by 2-amino-5-phosphonopentanoic acid (AP-5 (50-100µM)) indicating the involvement of NMDA receptors. Bicuculline (3-10 µM) enhanced the monosynaptic component suggesting electrically-evoked and/or glutamate induced GABA release inhibits the monosynaptic component via GABAA receptor activation. There were complex effects of bicuculline on polysynaptic components. In the mPFC both the mono- and polysynaptic components were attenuated by 5-HT (10-100 µM) and NA (30 and 60 µM) and the monosynaptic component was attenuated by DA (100 µM). In the OFC the mono- and polysynaptic components were also attenuated by 5-HT (100 µM), NA (10-100 µM) but DA (10-100 µM) had no effect. We propose that these pharmacologically characterised electrically-evoked field potentials in the mPFC and OFC are useful models for the study of prefrontal cortical physiology and pathophysiology., (Copyright © 2013 Elsevier B.V. and ECNP. All rights reserved.)

Serotonin (5-hydroxytryptamine, 5-HT) is involved in the regulation of feeding and digestion in many animals from worms to mammals. In insects, 5-HT functions both as a neurotransmitter and as a systemic hormone. Here we tested its role as a neurotransmitter in feeding and crop contractions and its role as a systemic hormone that affected feeding in adult foraging honeybees. We found 5-HT immunoreactive processes throughout the gut, including on the surface of the oesophagus, crop, proventriculus, and the midgut, as well as in the ventral nerve cord. mRNA transcripts for all four of the known bee 5-HT receptors (Am5-ht1A,2α,2β,7) were expressed in the crop and the midgut suggesting a functional role for 5-HT in these locations. Application of a cocktail of antagonists with activity against these known receptors to the entire gut in vivo reduced the rate of spontaneous contraction in the crop and proventriculus. Although feeding with sucrose caused a small elevation of endogenous 5-HT levels in the haemolymph, injection of exogenous 5-HT directly into the abdomen of the bee to elevate 5-HT in the haemolymph did not alter food intake. However, when 5-HT was injected into directly into the brain there was a reduction in intake of carbohydrate, amino acid, or toxin-laced food solutions. Our data demonstrate that 5-HT inhibits feeding in the brain and excites muscle contractions in the gut, but general elevation of 5-HT in the bee's haemolymph does not affect food intake., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A unique property of mitochondria in mammalian cells is their ability to physically interact and undergo dynamic events of fusion/fission that remodel their morphology and possibly their function. In cultured cells, metabolic perturbations similar to those incurred during exercise influence mitochondrial fusion and fission processes, but it is unknown whether exercise acutely alters mitochondrial morphology and/or membrane interactions in vivo. To study this question, we subjected mice to a 3-h voluntarily exercise intervention following their normal physical activity patterns, and quantified mitochondrial morphology and membrane interactions in the soleus using a quantitative electron microscopy approach. A single exercise bout effectively decreased blood glucose (P < 0.05) and intramyocellular lipid content (P < 0.01), indicating increased muscle metabolic demand. The number of mitochondria spanning Z-lines and proportion of electron-dense contact sites (EDCS) between adjacent mitochondrial membranes were increased immediately after exercise among both subsarcolemmal (+116%, P < 0.05) and intermyofibrillar mitochondria (+191%, P < 0.001), indicating increased physical interactions. Mitochondrial morphology, and abundance of the mitochondrial pro-fusion proteins Mfn2 and OPA1 were unchanged. Collectively, these results support the notion that mitochondrial membrane dynamics are actively remodelled in skeletal muscle, which may be regulated by contractile activity and the metabolic state. Future studies are required to understand the implications of mitochondrial dynamics in skeletal muscle physiology during exercise and inactivity.

The association of single-nucleotide polymorphisms (SNPs) in the human tryptophan hydroxylase 2 (TPH2) gene with anxiety traits and depression has been inconclusive. Observed inconsistencies might result from the fact that TPH2 polymorphisms have been studied in a genetically heterogeneous human population. A defined genetic background, control over environmental factors, and the ability to analyze the molecular and neurochemical consequences of introduced genetic alterations constitute major advantages of investigating SNPs in inbred laboratory mouse strains. To investigate the behavioral and neurochemical consequences of a functional C1473G SNP in the mouse Tph2 gene, we generated congenic C57BL/6N mice homozygous for the Tph2 1473G allele. The Arg(447) substitution in the TPH2 enzyme resulted in a significant reduction of the brain serotonin (5-HT) in vivo synthesis rate. Despite decreased 5-HT synthesis, we could detect neither a reduction of brain region-specific 5-HT concentrations nor changes in baseline and stress-induced 5-HT release using a microdialysis approach. However, using a [(35)S]GTP-γ-S binding assay and 5-HT(1A) receptor autoradiography, a functional desensitization of 5-HT(1A) autoreceptors could be identified. Furthermore, behavioral analysis revealed a distinct anxiety phenotype in homozygous Tph2 1473G mice, which could be reversed with chronic escitalopram treatment. Alterations in depressive-like behavior could not be detected under baseline conditions or after chronic mild stress. These findings provide evidence for an involvement of functional Tph2 polymorphisms in anxiety-related behaviors, which are likely not caused directly by alterations in 5-HT content or release but are rather due to compensatory changes during development involving functional desensitization of 5-HT(1A) autoreceptors.

The ascending 5-HT system has been and continues to be the subject of much research. The majority of in vivo electrophysiological and neurochemical studies of 5-HT function in rodents have been conducted in animals under anaesthesia - usually chloral hydrate or urethane. However, the effects of anaesthetics, on 5-HT function have not been systematically investigated. Here we used in vitro electrophysiology in dorsal raphe slices, to determine the effects of anaesthetically relevant concentrations of chloral hydrate (100 μM and 1 mM), urethane (10 and 30 mM), pentobarbitone (10 and 100 μM) and ketamine (10, 100 and 300 μM) on regulators of 5-HT firing activity. We examined i) basal firing (driven by α(1) adrenoceptors), ii) the excitatory response to N-methyl-d-aspartate (NMDA), iii) the 5-HT(1A) autoreceptor-mediated inhibitory response to 5-HT and iv) the GABA(A) receptor-mediated inhibitory response to 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridinyl-3-ol (THIP, gaboxadol). Pentobarbitone selectively enhanced the response to THIP. Ketamine decreased basal firing, attenuated the response to NMDA, and enhanced responses to both 5-HT and THIP. Chloral hydrate had marginal effects on basal firing, slightly attenuated the NMDA response, and enhanced both the 5-HT and THIP responses. Urethane increased basal firing, decreased the NMDA response, increased the response to THIP, but had no effect on the 5-HT response. Our data indicate that all anaesthetics tested significantly affect the regulators of 5-HT neuronal function. These findings will aid in the interpretation of previous reports of in vivo studies of the 5-HT system and will allow researchers to make a rational selection of anaesthetic for future studies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Whether animals experience human-like emotions is controversial and of immense societal concern [1-3]. Because animals cannot provide subjective reports of how they feel, emotional state can only be inferred using physiological, cognitive, and behavioral measures [4-8]. In humans, negative feelings are reliably correlated with pessimistic cognitive biases, defined as the increased expectation of bad outcomes [9-11]. Recently, mammals [12-16] and birds [17-20] with poor welfare have also been found to display pessimistic-like decision making, but cognitive biases have not thus far been explored in invertebrates. Here, we ask whether honeybees display a pessimistic cognitive bias when they are subjected to an anxiety-like state induced by vigorous shaking designed to simulate a predatory attack. We show for the first time that agitated bees are more likely to classify ambiguous stimuli as predicting punishment. Shaken bees also have lower levels of hemolymph dopamine, octopamine, and serotonin. In demonstrating state-dependent modulation of categorization in bees, and thereby a cognitive component of emotion, we show that the bees' response to a negatively valenced event has more in common with that of vertebrates than previously thought. This finding reinforces the use of cognitive bias as a measure of negative emotional states across species and suggests that honeybees could be regarded as exhibiting emotions., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Patients with bipolar disorder have abnormalities in glucocorticoid secretion, dopaminergic neurotransmission, and prefrontal cortical function. We hypothesized that the flattening of the diurnal glucocorticoid rhythm, commonly seen in bipolar disorder, modulates dopaminergic neurotransmission in the prefrontal cortex (PFC) leading to abnormalities in prefrontally mediated neurocognitive functions. To address this hypothesis, we investigated the effects of a flattened glucocorticoid rhythm on (i) the release of dopamine in the PFC and (ii) the transcription of genes in the ventral tegmental area (VTA) coding for proteins involved in presynaptic aspects of dopaminergic neurotransmission. Male rats were treated for 13-15 days with corticosterone (50 microg/ml in the drinking water) or vehicle (0.5% ethanol). Corticosterone treatment resulted in marked adrenal atrophy and flattening of the glucocorticoid rhythm as measured by repeated blood sampling. Animals treated with corticosterone showed markedly enhanced basal dopamine release in the PFC as measured by microdialysis in the presence of a dopamine reuptake inhibitor. Depolarization-evoked release was also enhanced, suggesting that the corticosterone effect on basal release did not result from an increase in the neuronal firing rate. Local blockade of terminal D(2) autoreceptors failed to normalize release to control values, suggesting that the enhanced release was not because of reduced autoreceptor sensitivity. In situ hybridization histochemistry showed that mRNAs coding tyrosine hydroxylase and the vesicular monoamine transporter 2 were elevated in the VTA of corticosterone-treated rats. Our data show that flattening of the glucocorticoid rhythm increases dopamine release in the PFC possibly as a result of increased synthesis and vesicular storage. This provides a mechanistic explanation for prefrontal dysfunction in bipolar and other affective disorders associated with glucocorticoid dysrhythmia.

The effects of combined treatment with a glucocorticoid receptor (GR) antagonist, Org 34850, and a selective serotonin reuptake inhibitor (SSRI), fluoxetine, were investigated on pre- and postsynaptic aspects of 5-HT neurotransmission. Rats were treated for 14 days with Org 34850 (15 mg per kg per day subcutaneously), fluoxetine (10 mg per kg per day intraperitoneally), or a combination of both drugs. [(3)H]-citalopram binding (an index of 5-HT transporter (5-HTT) expression) was only slightly affected by Org 34850 alone: decreased in cortex and midbrain and increased in hippocampus. In contrast, chronic fluoxetine markedly decreased 5-HTT levels in all regions. Importantly, this decrease was significantly enhanced by combined Org 34850/fluoxetine treatment. There were no changes in the expression of 5-HTT mRNA, suggesting these effects were not due to changes in gene transcription. Expression of tryptophan hydroxylase mRNA and both 5-HT(1A) autoreceptor mRNA and protein (assessed using [(3)H]-8-OH-DPAT binding) were unchanged by any treatment. The expression of postsynaptic 5-HT(1A) receptor protein in the forebrain was unaltered by fluoxetine, Org 34850 or the combined Org 34850/fluoxetine treatment. This downregulation of 5-HTT by fluoxetine and its enhancement by Org 34850 can explain our recent observation that GR antagonists augment the SSRI-induced increase in extracellular 5-HT. In addition, these data suggest that the augmentation of forebrain 5-HT does not result in downregulation of forebrain 5-HT(1A) receptor expression. Given the importance of 5-HT(1A) receptor-mediated transmission in the forebrain to the antidepressant response, these data indicate that co-administration of GR antagonists may be effective in augmenting the antidepressant response to SSRI treatment.

Dysregulation of mesolimbic dopamine (DA) neurotransmission has been implicated in bipolar disorder. DA release in the nucleus accumbens is reduced in rats treated chronically with the mood stabiliser lithium, and this effect is maintained for 3 days after withdrawal from the lithium treatment. We tested whether this decrease in DA release is due to an increase in D(2/3) autoreceptor sensitivity. In vivo microdialysis studies showed that in the shell of the nucleus accumbens, dialysate DA was decreased following chronic lithium treatment and 3 days after withdrawal from lithium treatment. The elevation of dialysate DA induced by local blockade of the terminal D(2/3) receptor was reduced in both lithium treated and lithium withdrawn groups. In vitro electrophysiology studies showed that chronic lithium treatment (and lithium withdrawal) did not alter either basal firing rate of DA neurones in the ventral tegmental area, or somatodendritic D(2/3) autoreceptor-mediated inhibition of firing. D(2) mRNA expression in the ventral tegmental area was unchanged by lithium treatment and lithium withdrawal. Our data suggest that the decrease in dopamine release in the nucleus accumbens induced by chronic lithium treatment is not the result of increased terminal or somatodendritic autoreceptor sensitivity or decreased firing rate of DA neurones.

Background: Selective serotonin reuptake inhibitor (SSRI) antidepressant drugs have a delayed onset and commonly produce an incomplete therapeutic response. The therapeutic actions of SSRIs are thought to depend on increased forebrain extracellular serotonin (5-HT), after desensitization of somatodendritic 5-HT(1A) autoreceptors. Here we determined whether concurrent glucocorticoid receptor (GR) blockade enhances these neurochemical responses to the SSRI fluoxetine., Methods: Male rats were treated (3, 7, or 14 days) with either fluoxetine (10 mg/kg IP) or vehicle once daily, in combination with either a GR antagonist (Org 34850 15 mg/kg SC or Org 34517 25 mg/kg SC) or vehicle twice daily. After treatment, 5-HT in the medial prefrontal cortex was measured by microdialysis., Results: Chronic fluoxetine treatment (14 days) raised basal 5-HT and also attenuated the fall in 5-HT after acute systemic administration of fluoxetine (10 mg/kg IP), indicating desensitization of 5-HT(1A) autoreceptors. Concurrent chronic administration (14 days) of Org 34850 or Org 34517 enhanced the fluoxetine-induced increase in basal 5-HT. Org 34850 also hastened the 5-HT(1A) autoreceptor desensitization induced by chronic fluoxetine treatment. Org 34850 alone (14 days) failed to alter basal 5-HT or 5-HT(1A) autoreceptor desensitization., Conclusions: Antidepressant response is proposed to depend on 5-HT(1A) autoreceptor desensitization and elevation of forebrain 5-HT. These data suggest adjunctive GR antagonists might both hasten and enhance antidepressant responses to SSRIs.

The glutamatergic regulation of 5-hydroxytryptamine (5-HT) neuronal activity has not been extensively studied. Here, we used extracellular single unit recording in midbrain slices to examine glutamate receptor mediated effects on 5-HT neuronal activity in the dorsal raphe nucleus (DRN) and the median raphe nucleus (MRN). Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA; 1 and 3 microm) concentration-dependently increased firing in 5-HT neurons in both the DRN and the MRN. The response to AMPA was blocked by the AMPA receptor antagonist, 6,7-dinitroquinoxaline-2,3(1H-4H)-dione (DNQX; 10 microm) but not the N-methyl-d-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonopentanoic acid (AP-5; 50 microm). NMDA (10-100 microm) also increased 5-HT neuronal firing in a concentration-dependent manner in both the DRN and MRN; a response that was blocked by AP-5 (50 microm). In some DRN neurons the NMDA response was partially antagonized by DNQX (10 microm) suggesting that NMDA, as well as directly activating 5-HT neurons, evokes local release of glutamate, which indirectly activates AMPA receptors on 5-HT neurons. Responses of DRN 5-HT neurons to AMPA and NMDA were enhanced by the gamma-amino-butyric acid (GABA)(A) receptor antagonist, bicuculline (50 microm), suggesting that both AMPA and NMDA increase local release of GABA. Finally in the DRN the 5-HT(1A) receptor antagonist, WAY100635 (100 nm), failed to enhance the response of 5-HT neurons to AMPA and caused only a small increase in the excitatory response to NMDA suggesting a low degree of tonic activation of 5-HT(1A) autoreceptors even when 5-HT neuronal firing rate is high.

The dorsal raphe nucleus (DRN) is the origin of much of the 5-HT innervation of the forebrain. The activity of DRN 5-HT neurons is regulated by a number of receptors including GABA(A) and 5-HT(1A) inhibitory receptors and by excitatory alpha(1)-adrenoceptors. Using in vitro electrophysiological recording we investigated the action of progesterone and its metabolite, allopregnanolone on receptor-mediated responses of DRN 5-HT neurons. Neither allopregnanolone nor progesterone affected the alpha(1)-adrenoceptor agonist-induced firing. Allopregnanolone also had no effect on the inhibitory response to 5-HT. However, allopregnanolone significantly potentiated the inhibitory responses to GABA(A) receptor agonists. Progesterone did not enhance GABA(A) receptor-meditated inhibitory responses. Thus, the neuroactive metabolite of progesterone, allopregnanolone, has the ability to cause potentiation of GABA(A)-mediated inhibition of DRN 5-HT neurons. This effect on 5-HT neurotransmission may have relevance for mood disorders commonly associated with reproductive hormone events, such as premenstrual dysphoric disorder and postpartum depression.

5-HT neurones in the median raphe nucleus (MRN) are involved in anxiety and the sleep/wake cycle. Here, using in vitro electrophysiology, we examined if the firing of MRN 5-HT neurones is regulated by GABA(A) receptors. The GABA(A) receptor agonists THIP and muscimol caused concentration dependent inhibition of MRN 5-HT neurones. The GABA(A) receptor antagonist bicuculline blocked the responses to THIP and muscimol. Bicuculline alone increased the basal firing activity. Responses to THIP were enhanced by the Z hypnotic zolpidem at concentrations selective for the alpha(2)/alpha(3) subunits of the GABA(A) receptor (0.2 and 1microM) but not at a concentration selective for the alpha(1) subunit (0.02microM). Consistent with these functional data, 5-HT neurones have been shown to express the alpha(3) (but not alpha(2)) subunit. The anxiolytic effects of GABA(A) receptor modulators are reportedly mediated by alpha(3)-containing receptors. Hence the MRN 5-HT system may be a target for anxiolytic drugs.

The median raphe nucleus and dorsal raphe nucleus together are the major source of ascending 5-HT projections. Here, using in vitro extracellular single unit electrophysiology we examined the responses of individual neurones in the rat median raphe nucleus and dorsal raphe nucleus to alpha(1)-adrenoceptor and 5-HT(1A) receptor activation and made comparisons between the two nuclei. In the presence of the alpha(1)-adrenoceptor agonist phenylephrine (1microM) all spontaneously active neurones recorded in the median and dorsal raphe nuclei fired slowly (<5Hz) and regularly. Most were inhibited by 5-HT (10-50microM), although a few were excited by 5-HT. 5-HT-induced inhibition was attenuated by the 5-HT(1A) receptor antagonist, WAY100635 (100nM). Compared to those in the dorsal raphe nucleus, the neurones in the median raphe nucleus which were inhibited by 5-HT had: (1) lower basal firing rates in the continuous presence of phenylephrine (1microM), (2) smaller excitatory responses to higher concentrations of phenylephrine (3-10microM), (3) smaller excitatory responses to brief application of norepinephrine (10-100microM) and (4) smaller inhibitory responses to 5-HT (10-50microM). The lower sensitivity of median raphe neurones to alpha(1)-adrenoceptor excitation and 5-HT(1A) receptor inhibition will have consequences for 5-HT neurotransmission in forebrain regions innervated by the two nuclei.

The therapeutic mechanism of action of lithium in the treatment of bipolar disorder is not well understood. Dysfunction of both 5-HT(1A) receptor mediated neurotransmission and the glucocorticoid receptor is associated with mood disorders, and preclinical studies suggest that lithium treatment can modulate these receptor subtypes. In this study, we investigated the effect of chronic lithium treatment on 5-HT(1A) receptors and glucocorticoid receptors in the rat brain. Male Sprague-Dawley rats were treated with lithium (3 mmol/kg/day) or saline for 28 days via subcutaneous implanted mini-osmotic pumps. After 28 days of treatment, the expression of mRNA for 5-HT(1A) receptors and glucocorticoid receptors in the rat hippocampus and dorsal raphe nucleus was determined by in situ hybridization histochemistry. Chronic administration of lithium decreased mRNA coding for post-synaptic 5-HT(1A) receptors in hippocampal subregions but not for somatodentritic 5-HT(1A) receptors in the dorsal raphe nucleus. Chronic administration of lithium did not affect mRNA coding for glucocorticoid receptors in hippocampal subregions or in the dorsal raphe nucleus. Mean plasma lithium levels in the lithium-treated group were 0.50 +/- 0.03 mmol/l; all animals appeared healthy and maintained a normal increase in body weight. Given recent reports implicating hypersensitive post-synaptic 5-HT(1A) receptors in bipolar manic patients, the present study suggests that down-regulation of this receptor population may be important in the therapeutic mechanism of action of lithium.

Evidence from electrophysiological studies suggests that 5-HT neuronal firing in the dorsal raphe nucleus (DRN) may be regulated by both GABA(A) and GABA(B) receptors. Here, we addressed the question of whether the activity of individual 5-HT neurons is regulated by both GABA(A) and GABA(B) receptors. In addition, we examined the concentration-response relationships of GABA(A) and GABA(B) receptor activation and determined if GABA receptor regulation of 5-HT neuronal firing is altered by moderate alterations in circulating corticosterone. The activity of 5-HT neurons in the DRN of the rat was examined using in vitro extracellular electrophysiology. The firing of all individual neurons tested was inhibited by both the GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridin-3-ol hydrochloride (THIP) (25 microM) and the GABA(B) receptor agonist baclofen (1 microM). Responses to THIP (5, 10, 25 microM) and baclofen (1, 3, 10 microM) were concentration dependent and attenuated by the GABA(A) and GABA(B) receptor antagonists, bicuculline (50 microM) and phaclofen (200 microM), respectively. To examine the effects of corticosterone on the sensitivity of 5-HT neurons to GABA receptor activation, experiments were conducted on adrenalectomized animals with corticosterone maintained for two weeks at either a low or moderate level within the normal diurnal range. These changes in corticosterone levels had no significant effects on the 5-HT neuronal response to either GABA(A) or GABA(B) receptor activation. The data indicate that the control of 5-HT neuronal activity by GABA is mediated by both GABA(A) and GABA(B) receptors and that this control is insensitive to moderate changes in circulating glucocorticoid levels.

Circulating glucocorticoid levels vary with stress and psychiatric illness and play a potentially important role in regulating transmitter systems that regulate mood. To determine whether chronic variation in corticosterone levels within the normal diurnal range altered the control of 5-hydroxytryptamine (5-HT) neuronal activity, male rats were adrenalectomized and implanted with either a 2% or 70% corticosterone/cholesterol pellet (100 mg). Two weeks later, the regulation of 5-HT neuronal activity in the dorsal raphe nucleus was studied by in vitro electrophysiology. At this time, serum corticosterone levels approximated the low-point (2%) and mid-point (70%) of the diurnal range. The excitatory response of 5-HT neurones to the alpha1-adrenoceptor agonist phenylephrine (1-11 microM) was significantly greater in the 2% group compared to the 70% group. By contrast, the inhibitory response to 5-HT (10-50 microM) was significantly lower in the 2% group compared to the 70% group. Thus, chronic variation in circulating corticosterone over a narrow part of the normal diurnal range causes a shift in the balance of positive and negative regulation of 5-HT neurones, with increased alpha 1-adrenoceptor-mediated excitation and reduced 5-HT-mediated autoinhibition at lower corticosterone levels. This shift would have a major impact on control of 5-HT neuronal activity.

Both glucocorticoids and selective serotonin reuptake inhibitors (SSRIs) alter aspects of 5-HT function including somatodendritic 5-HT1A autoreceptor sensitivity. Many depressed patients prescribed SSRIs have pre-existing flattened diurnal gluococorticoid rhythm. In these patients, interactions between flattened glucocorticoid rhythm and chronic SSRIs, which impact on the SSRI's ability to elevate forebrain 5-HT, may alter clinical efficacy. To address this issue rats underwent implantation of slow-release corticosterone (75 mg pellet s.c.) (to flatten the glucocorticoid rhythm) or sham surgery, and injection of fluoxetine (10 mg/kg/day i.p., 12 days) or vehicle. Using microdialysis in the frontal cortex we found that (21 h after the last injection) extracellular 5-HT was elevated in fluoxetine- or corticosterone-treated animals, but not in those treated with corticosterone plus fluoxetine. In fluoxetine-treated animals, blockade of terminal reuptake by local perfusion of fluoxetine increased 5-HT to the same level as it did in controls, suggesting normal terminal 5-HT release after chronic fluoxetine. However, 5-HT levels following local reuptake blockade in both the corticosterone and corticosterone plus fluoxetine groups were lower than controls, suggesting a corticosterone-induced decrease in terminal release. Finally in fluoxetine, corticosterone, and corticosterone plus fluoxetine groups, there was marked 5-HT1A receptor desensitization, evidenced by attenuation of the decrease in 5-HT release following systemic fluoxetine injection. The data indicate that, despite desensitization of 5-HT1A autoreceptors, concurrent flattened glucocorticoid rhythm compromises the ability of SSRIs to elevate forebrain 5-HT. These findings suggest a potential mechanism for the reduced antidepressant efficacy of SSRIs in those patients with pre-existing glucocorticoid abnormalities.

The ventral part of the medial prefrontal cortex (mPFC) plays an important role in mood and cognition. This study examined the effect of the 5-HT in this region by measuring the electrophysiological response of ventral mPFC neurones to electrical stimulation of the dorsal and median raphe nuclei (DRN and MRN), which are the source of the 5-HT input. DRN or MRN stimulation evoked a consistent, short-latency, post-stimulus inhibition in the majority of ventral mPFC neurones tested (DRN: 44/73 neurones; MRN: 24/31 neurones). Some neurones responded to DRN or MRN stimulation with antidromic spikes indicating that they were mPFC-raphe projection neurones. Both DRN- and MRN-evoked inhibitions were attenuated by systemic administration of the 5-HT1A antagonist WAY 100635 (0.1 mg/kg i.v.). DRN-evoked inhibition was also attenuated by iontophoretic application of WAY 100635 and by systemic administration of the 5-HT1A antagonist, NAD-299 (4 mg/kg i.v.) but not the 5-HT2 antagonist ketanserin (4 mg/kg, i.v.). These data suggest that DRN and MRN 5-HT neurones inhibit neurones in the ventral mPFC via activation of 5-HT1A receptors. Some of these mPFC neurones may be part of a 5-HT1A receptor-controlled postsynaptic feedback loop to the DRN and MRN.

Early life adversity is associated with an increased incidence of psychiatric illness in adulthood. Although the mechanisms underlying this association are unclear, one possible substrate is brain 5-hydroxytryptamine neurotransmission, which is reportedly abnormal in several psychiatric disorders. This study examined the effect of a rat model of early life adversity, early maternal separation, on 5-hydroxytryptamine neurotransmission in adulthood. In vitro electrophysiological experiments revealed that, in early maternal separation rats compared with controls, the sensitivity of alpha1-adrenoceptors on 5-hydroxytryptamine neurons in the dorsal raphe nucleus was significantly reduced, whilst the sensitivity of 5-hydroxytryptamine1A receptors showed a nonsignificant trend to reduction. In in vivo microdialysis experiments, the 5-hydroxytryptamine1A receptor agonist-induced suppression of 5-hydroxytryptamine release in the frontal cortex was reduced in early maternal separation animals, suggesting desensitization of 5-hydroxytryptamine1A autoreceptors. There was no increase in basal 5-hydroxytryptamine in the frontal cortex as measured by microdialysis and a nonsignificant trend towards increased basal firing activity of classical (non-bursting) 5-hydroxytryptamine neurons in the dorsal raphe nucleus measured by in vivo electrophysiology. Finally, early maternal separation failed to alter expression of messenger ribonucleic acids coding for 5-hydroxytryptamine1A or alpha1B receptors in the dorsal raphe nucleus as measured by in situ hybridization histochemistry, suggesting that functional changes in receptor sensitivity observed are not due to changes in receptor gene transcription. The findings demonstrate that early life adversity programs changes in sensitivity of the two principal regulators of 5-hydroxytryptamine neuronal activity. Similar effects in humans may contribute to the increased incidence of psychiatric illness in individuals exposed to early life adversity.

Subtle changes in glucocorticoid levels, including a flattening of the diurnal rhythm with raised nadir, are prevalent, being characteristic of both aging and major depression. Both these conditions are also associated with deficits in hippocampally mediated cognitive functions. We hypothesized that this profile of glucocorticoid levels causes structural and functional changes in the hippocampus, which in turn may engender cognitive deficits. We implanted slow-release corticosterone pellets into adrenally intact adult male rats to produce a flattened glucocorticoid rhythm with levels clamped midway between the normal nadir and zenith. Using density profile analysis we measured hippocampal expression of messenger RNAs encoding structural and functional proteins. In rats with a flattened glucocorticoid rhythm, the expression of the mRNA coding for microtubule associated protein-2b (MAP2b) was reduced in CA3 relative to sham-operated controls, but unchanged in dentate gyrus and CA1. In contrast, the expression of the mRNA coding the alpha subunit of calcium-calmodulin dependent kinase (CAMKIIalpha) was reduced in dentate gyrus in animals with a flattened glucocorticoid rhythm, but unchanged in CA3. The expression of the mRNA coding the synaptic vesicle protein synaptophysin was unchanged in both CA3 and dentate gyrus. The data indicate that a flattening of the normal diurnal glucocorticoid rhythm decreases the hippocampal expression of mRNAs coding key structural and functional proteins, and does so in a regionally selective manner. The data may have relevance for cognitive deficits characteristic of aging and depression.

Depression is associated with glucocorticoid abnormalities, in particular a flattening of the diurnal cortisol rhythm. Recent data suggest that an important factor in the aetiology of depression may be a deficit in the function and expression of 5-HT(1A) receptors, which has been reported in depressed patients. The present study assessed the possibility that this cortisol abnormality is causal in the 5-HT(1A) receptor deficits. First, a rat model of flattened glucocorticoid rhythm was developed. Controlled release corticosterone pellets implanted for 14 days flattened the corticosterone rhythm and maintained levels constant midway between the nadir and zenith levels observed in sham-operated rats. Secondly, using microdialysis to assess 5-HT release in the hippocampus, the inhibitory response to 8-OHDPAT was measured to determine the sensitivity of somatodendritic 5-HT(1A) autoreceptors. Corticosterone treatment was found to induce a significant attenuation in the response to 8-OHDPAT, indicating functional desensitization of somatodendritic 5-HT(1A) autoreceptors. There was no effect of corticosterone treatment on basal extracellular 5-HT levels. The data suggest that the glucocorticoid abnormalities associated with depression may impact on the functioning of 5-HT(1A) receptors in the brain. These findings suggest that resolution of cortisol abnormalities may be a valuable target for pharmacotherapy in the treatment of depression.

5-HT(1A)-mediated autoinhibition of neurones in the dorsal raphe nucleus (DRN) is considered to be the principal inhibitory regulator of 5-HT neuronal activity. The activation of this receptor by endogenous 5-HT was investigated using electrophysiological recordings from the rat DRN in vitro. At a concentration which blocked the inhibitory effect of exogenous 5-HT, the 5-HT(1A) antagonist WAY 100635 did not alter basal firing rate or modulate the excitatory response to the alpha(1)-agonist phenylephrine. Blockade of 5-HT reuptake by a concentration of fluoxetine, which enhanced the inhibitory effect of exogenous 5-HT, lowered phenylephrine-induced basal firing presumably due to potentiation of the effect of endogenous 5-HT. However, this effect was not firing rate dependent and neither the proportional increase nor the time-course of the response to a higher concentration of phenylephrine were altered in the presence of fluoxetine. These data suggest that the inhibitory 5-HT(1A) receptor on raphe neurones is neither tonically activated nor plays any role in modulating the response to excitatory transmitters. Thus, at physiological firing rates this receptor does not appear to function as an autoreceptor of serotonergic neurones of the DRN.

We have previously described a population of 5-hydroxytryptamine neurons which repetitively fires bursts of usually two (but occasionally three or four) action potentials, with a short (<20 ms) interspike interval within a regular low-frequency firing pattern. Here we used a paradigm of electrical stimulation comprising twin pulses (with 7- or 10-ms inter-pulse intervals) to mimic this burst firing pattern, and compared the effects of single- and twin-pulse electrical stimulations in models of pre- and postsynaptic 5-hydroxytryptamine function. Firstly, we measured the effect of direct electrical stimulation (2 Hz for 2 min) of rat brain slices on efflux of preloaded [3H]5-hydroxytryptamine. In this in vitro model, twin-pulse stimulation increased the efflux of tritium by about twice as much as did single-pulse stimulation. This effect was evident in the medial prefrontal cortex (area under the curve: 2. 59+/-0.34 vs 1.28+/-0.22% relative fractional release), as well as in the caudate-putamen (3.93+/-0.65 vs 2.17+/-0.51%) and midbrain raphe nuclei (5.42+/-1.05 vs 2.51+/-0.75%). Secondly, we used in vivo microdialysis to monitor changes in endogenous extracellular 5-hydroxytryptamine in rat medial prefrontal cortex in response to electrical stimulation (3 Hz for 10 min) of the dorsal raphe nucleus. In this model, twin-pulse stimulation of the dorsal raphe nucleus increased 5-hydroxytryptamine by approximately twice as much as did single-pulse stimulation at the same frequency (area under the curve: 50.4+/-9.0 vs 24.2+/-4.4 fmol). Finally, we used in vivo extracellular recording to follow the response of postsynaptic neurons in the rat medial prefrontal cortex to 5-hydroxytryptamine released by dorsal raphe stimulation. Electrical stimulation of the dorsal raphe nucleus (1 Hz) induced a clear-cut poststimulus inhibition in the majority of cortical neurons tested. In these experiments, the duration of poststimulus inhibition following twin-pulse stimulation was markedly longer than that induced by single-pulse stimulation (200+/-21 vs 77+/-18.5 ms). Taken together, the present in vitro and in vivo data suggest that in 5-hydroxytryptamine neurons, short bursts of action potentials will propagate along the axon to the nerve terminal and will enhance both the release of 5-hydroxytryptamine and its postsynaptic effect.

The antidepressant efficacy of selective serotonin reuptake inhibitors (SSRIs) might be enhanced by co-administration of 5-HT1A receptor antagonists. Thus, we have recently shown that the selective 5-HT1A receptor antagonist, WAY 100635, blocks the inhibitory effect of an SSRI on 5-HT cell firing, and enhances its ability to elevate extracellular 5-HT in the forebrain. Here we determined whether the beta-adrenoceptor/5-HT1A receptor ligands (+/-)-pindolol, (-)-tertatolol and (-)-penbutolol, interact with paroxetine in a similar manner. Both (-)-tertatolol (2.4 mg kg(-1) i.v.) and (-)-penbutolol (2.4 mg kg(-1) i.v.) enhanced the effect of paroxetine (0.8 mg kg(-1) i.v.) on extracellular 5-HT in the frontal cortex, whilst (+/-)-pindolol (4 mg kg(-1) i.v.) did not. (-)-Tertatolol (2.4 mg kg(-1) i.v.) alone caused a slight increase in 5-HT however, (-)-penbutolol (2.4 mg kg(-1) i.v.) alone had no effect. In electrophysiological studies (-)-tertatolol (2.4 mg kg(-1) i.v.) alone had no effect on 5-HT cell firing but blocked the inhibitory effect of paroxetine. In contrast, (-)-penbutolol (0.1-0.8 mg kg(-1) i.v.) itself inhibited 5-HT cell firing, and this effect was reversed by WAY 100635 (0.1 mg kg(-1) i.v.). We have recently shown that (+/-)-pindolol inhibits 5-HT cell firing via a WAY 100635-sensitive mechanism. Our data suggest that (-)-tertatolol enhances the effect of paroxetine on forebrain 5-HT via blockade of 5-HT1A autoreceptors which mediate paroxetine-induced inhibition of 5-HT cell firing. In comparison, the mechanisms by which (-)-penbutolol enhances the effect of paroxetine on extracellular 5-HT is unclear, since (-)-penbutolol itself appears to have agonist properties at the 5-HT1A autoreceptor. Indeed, the agonist action of (+/-)-pindolol at 5-HT1A autoreceptors probably explains its inability to enhance the effect of paroxetine on 5-HT in the frontal cortex. Overall, our data suggest that both (-)-tertatolol and (-)-penbutolol are superior to (+/-)-pindolol in terms of enhancing the effect of an SSRI on extracellular 5-HT. Both (-)-tertatolol and (-)-penbutolol are worthy of investigation for use as adjuncts to SSRIs in the treatment of major depression.

1. It has been hypothesized that 5-HT1A autoreceptor antagonists may enhance the therapeutic efficacy of SSRIs and other antidepressants. Although early clinical trials with the beta-adrenoceptor/5-HT1 ligand, pindolol, were promising, the results of recent more extensive trials have been contradictory. Here we investigated the actions of pindolol at the 5-HT1A autoreceptor by measuring its effect on 5-HT neuronal activity and release in the anaesthetized rat. 2. Pindolol inhibited the electrical activity of 5-HT neurones in the dorsal raphe nucleus (DRN). This effect was observed in the majority of neurones tested (10/16), was dose-related (0.2-1.0 mg kg(-1), i.v.), and was reversed by the 5-HT1A receptor antagonist, WAY 100635 (0.1 mg kg(-1), i.v.), in 6/7 cases tested. 3. Pindolol also inhibited 5-HT neuronal activity when applied microiontophoretically into the DRN in 9/10 neurones tested. This effect of pindolol was current-dependent and blocked by co-application of WAY 100635 (3/3 neurones tested). 4. In microdialysis experiments. pindolol caused a dose-related (0.8 and 4 mg kg(-1), i.v.) fall in 5-HT levels in dialysates from the frontal cortex (under conditions where the perfusion medium contained 1 microM citalopram). In rats pretreated with WAY 100635 (0.1 mg kg(-1), i.v.), pindolol (4 mg kg(-1), i.v.) did not decrease, but rather increased 5-HT levels. 5. We conclude that, under the experimental conditions used in this study, pindolol displays agonist effects at the 5-HT1A autoreceptor. These data are relevant to previous and ongoing clinical trials of pindolol in depression which are based on the rationale that the drug is an effective 5-HT1A autoreceptor antagonist.

It is proposed that 3,4-methylenedioxymethamphetamine (MDMA; Ecstasy) is more toxic to 5-HT neurones projecting from the dorsal raphe nucleus (DRN) than to those from the median raphe nucleus (MRN). Since increased 5-HT release has been associated with MDMA-induced neurotoxicity, MDMA may have a DRN-selective 5-HT releasing effect. Here we have compared the effects of acute MDMA on DRN and MRN 5-HT pathways using in vivo electrophysiological and neurochemical techniques. MDMA inhibited the firing of 5-HT neurones in both the DRN and the MRN, and did so with similar potency (ED50 values, 0.589 +/- 0.151 (8) and 0.588 +/- 0.207 (6) mg/kg i.v., respectively). In both nuclei this inhibitory effect was reversed by the selective 5-HT1A receptor antagonist, WAY 100635 (0.1 mg/kg i.v.). Microdialysis measurements were made in the frontal cortex and dorsal hippocampus, regions which receive a DRN- and an MRN-selective 5-HT innervation, respectively. A dose of 1 mg/kg i.v. MDMA increased extracellular 5-HT 3-fold in both the frontal cortex and dorsal hippocampus. A higher dose (3 mg/kg i.v.) increased 5-HT levels 8-fold in both regions. Overall, our data suggest that MDMA releases 5-HT from the cell body and terminal regions of both DRN and MRN 5-HT pathways, and does so in a qualitatively and quantitatively similar fashion. We conclude that any DRN-selectivity in the neurotoxic effects of MDMA is not due to a DRN-selective, acute 5-HT releasing action of the drug.

1. Selective 5-hydroxytryptamine (5-HT; serotonin) reuptake inhibitors (SSRIs) cause a greater increase in extracellular 5-HT in the forebrain when the somatodendritic 5-HT1A autoreceptor is blocked. Here, we investigated whether blockade of the terminal 5-HT1B autoreceptor influences a selective 5-HT reuptake inhibitor in the same way, and whether there is an additional effect of blocking both the 5-HT1A and 5-HT1B autoreceptors. 2. Extracellular 5-HT was measured in frontal cortex of the anaesthetized rat by use of brain microdialysis. In vivo extracellular recordings of 5-HT neuronal activity in the dorsal raphe nucleus (DRN) were also carried out. 3. The selective 5-HT reuptake inhibitor, paroxetine (0.8 mg kg-1, i.v.), increased extracellular 5-HT about 2 fold in rats pretreated with the 5-HT1A receptor antagonist, WAY100635. When administered alone neither paroxetine (0.8 mg kg-1, i.v.) nor WAY100635 (0.1 mg kg-1, i.v.) altered extracellular 5-HT levels. 4. Paroxetine (0.8 mg kg-1, i.v.) did not increase 5-HT in rats pretreated with the 5-HT1B/D receptor antagonist, GR127935 (1 mg kg-1, i.v.). GR127935 (1 and 5 mg kg-1, i.v.) had no effect on extracellular 5-HT when administered alone. 5. Interestingly, paroxetine (0.8 mg kg-1, i.v.) caused the greatest increase in 5-HT (up to 5 fold) when GR127935 (1 or 5 mg kg-1, i.v.) was administered in combination with WAY100635 (0.1 mg kg-1, i.v.). Administration of GR127935 (5 mg kg-1, i.v.) plus WAY100635 (0.1 mg kg-1, i.v.) without paroxetine, had no effect on extracellular 5-HT in the frontal cortex. 6. Despite the lack of effect of GR127935 on 5-HT under basal conditions, when 5-HT output was elevated about 3 fold (by adding 1 microM paroxetine to the perfusion medium), the drug caused a dose-related (1 and 5 mg kg-1, i.v.) increase in 5-HT. 7. By itself, GR127935 slightly but significantly decreased 5-HT cell firing in the DRN at higher doses (2.0-5.0 mg kg-1, i.v.), but did not prevent the inhibition of 5-HT cell firing induced by paroxetine. 8. In summary, our results suggest that selective 5-HT reuptake inhibitors may cause a large increase in 5-HT in the frontal cortex when 5-HT autoreceptors on both the somatodendrites (5-HT1A) and nerve terminals (5-HT1B) are blocked. This increase is greater than when either set of autoreceptors are blocked separately. The failure of a 5-HT1B receptor antagonist alone to enhance the effect of the selective 5-HT reuptake inhibitor in our experiments may be related to a lack of tone on the terminal 5-HT1B autoreceptor due to a continued inhibition of 5-HT cell firing. These results are discussed in relation to the use of 5-HT autoreceptor antagonists to augment the antidepressant effect of selective 5-HT reuptake inhibitors.

Improved clinical antidepressant efficacy may result if the acute inhibition of 5-HT cell firing induced by antidepressants is prevented. Here we examined whether inhibition of 5-HT cell firing by non-selective 5-HT uptake inhibiting antidepressant drugs is reversed by a selective 5-HT1A receptor antagonist. In addition, we examined whether concomitant blockade of NA uptake offsets the inhibition of 5-HT cell firing resulting from 5-HT uptake blockade. Antidepressants which block 5-HT uptake (paroxetine, clomipramine, amitriptyline, venlafaxine), all caused dose-dependent and complete inhibition of 5-HT cell firing. Desipramine, a selective NA uptake blocker, caused a slight reduction in firing. The selective 5-HT1A receptor antagonist, WAY 100635, reversed the inhibition of 5-HT cell firing induced by clomipramine, amitriptyline, venlafaxine, and paroxetine, but not that induced by the alpha 1 adrenoceptor antagonist, prazosin. Desipramine, at a dose which increased extracellular NA in the DRN, reversed the effect of prazosin but did not alter the ability of paroxetine to inhibit 5-HT cell firing. Our data indicate that antidepressant drugs with 5-HT uptake blocking properties inhibit 5-HT cell firing via activation of 5-HT1A autoreceptors, and do so irrespective of their effects on NA uptake. These data are discussed in relation to the application of 5-HT1A receptor antagonists to enhance the clinical efficacy of antidepressant drugs.

We report the effects of the monoamine oxidase inhibitor, tranylcypromine, combined with the 5-HT1A receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl cyclohexanecarboxamide (WAY 100635), on both 5-hydroxytryptamine (5-HT) cell firing and cortical extracellular 5-HT in the rat. Tranylcypromine inhibited 5-HT cell firing in the dorsal raphe nucleus dose-dependently (ED50 5 mg/kg i.v.). In microdialysis experiments, tranylcypromine (5 mg/kg i.v.) increased extracellular 5-HT in the frontal cortex. WAY 100635 (0.1 mg/kg i.v.) both reversed the inhibition of 5-HT cell firing and facilitated the increase in extracellular 5-HT. In conclusion, WAY 100635 enhances the effect of tranylcypromine on presynaptic 5-HT function. These data are relevant to clinical evidence that co-therapy with a 5-HT1A receptor antagonist improves the antidepressant efficacy of a monoamine oxidase inhibitor.

In experimental animals, administration of 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) leads to extensive, but incomplete, loss of 5-hydroxytryptamine (5-HT) innervation in the brain. Here, we report the effects of MDMA on 5-HT neuronal function measured in the rat in vivo using electrophysiological and microdialysis techniques. Two weeks after administration of an established neurotoxic regimen of MDMA (20 mg/kg s.c., twice daily for 4 days) we found; 1) no change in either the density or the firing activity of 5-HT neurons in the dorsal raphé nucleus; 2) no change in basal extracellular 5-HT in either the frontal cortex or the hippocampus, although extracellular 5-hydroxyindoleacetic acid was reduced by about 50% in both regions; and 3) no change in the amount of 5-HT released in the hippocampus in response to electrical stimulation (5 Hz) of either the dorsal or medial raphé nucleus, but a marked reduction in the amount of 5-HT released in the frontal cortex after electrical stimulation of the dorsal raphé nucleus. In summary, although MDMA causes marked 5-HT neurotoxicity, our data suggest that 5-HT cell firing is unchanged and, furthermore, that 5-HT release is maintained in some (but not all) forebrain regions even in response to physiological levels of stimulation.

Previous electrophysiological studies have shown that spontaneously active mesencephalic 5-hydroxytryptaminergic neurons of anaesthetized or freely moving animals fire solitary spikes in a slow, regular pattern. In the present study, using extracellular single unit recordings from dorsal and median raphe neurons of the anaesthetized rat, an additional electrophysiological property of a sub-population of presumed 5-hydroxytryptaminergic neurons was observed. These neurons, during their otherwise regular firing pattern, repeatedly fired two (or occasionally three or even four) spikes where only one was expected. Spikes in this burst-like repetitive firing mode (spikes in doublets or triplets) occurred in a short time interval (range: 2.4-11.5 ms), and with a diminishing spike amplitude. Cross-correlation analysis of spikes in doublets revealed a very high interdependency between them. The proportion of spikes in doublets to solitary spikes showed great variation between different neurons, ranging from 5 to 95% of the total spikes displayed. However, for each neuron the proportion of spikes in doublets to solitary spikes, and the time interval between the spikes in doublets, remained constant during control recordings. All these features are characteristic of single neurons firing in a repetitive firing pattern rather than simultaneous recordings of two separate 5-hydroxytryptaminergic neurons. Repetitive firing neurons were recorded with a similar frequency in both chloral hydrate and Saffan anaesthetized rats, and were detected using both glass and metal electrodes. Furthermore, neurons with a repetitive firing pattern were inhibited by intravenous administration of a selective 5-hydroxytryptamine1A receptor agonist and a 5-hydroxytryptamine reuptake inhibitor, thus displaying responses typical of 5-hydroxytryptaminergic neurons. Repetitive firing neurons occurred in both the dorsal and median raphe nuclei, although they were much more frequent in the dorsal raphe nucleus (91 of 332 neurons). The occurrence of repetitive firing neurons in the midbrain raphe nuclei is a newly described phenomenon which may indicate unique properties of a sub-population of 5-hydroxytryptaminergic neurons. In functional terms, it could modify both axonal and dendritic 5-hydroxytryptamine release, and provide an additional option for neuronal information signalling.