Multiple Nonauditory Cortical Regions Innervate the Auditory Midbrain.

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.

Puncta of Neuronal Nitric Oxide Synthase (nNOS) Mediate NMDA Receptor Signaling in the Auditory Midbrain.

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.

A role for nitric oxide in serotonin neurons of the midbrain raphe nuclei.

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.

Electrical stimulation of the ventral tegmental area evokes sleep-like state transitions under urethane anaesthesia in the rat medial prefrontal cortex via dopamine D1 -like receptors.

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 D1 -like receptor activation as it was almost completely blocked by systemic administration of the D1 -like receptor antagonist SCH23390. Overall, our data demonstrate that activation of dopamine D1 -like receptors in the mPFC is important for regulating sleep-like state transitions.

Dorsal vs. ventral differences in fast Up-state-associated oscillations in the medial prefrontal cortex of the urethane-anesthetized rat.

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.

Motor, somatosensory, and executive cortical areas elicit monosynaptic and polysynaptic neuronal activity in the auditory midbrain.

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.

Nitric oxide signalling underlies salicylate-induced increases in neuronal firing in the inferior colliculus: A central mechanism of tinnitus?

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.

Effect of chronic lithium treatment on D2/3 autoreceptor regulation of dopaminergic function in the rat.

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.

Glucocorticoid receptor antagonists hasten and augment neurochemical responses to a selective serotonin reuptake inhibitor antidepressant.

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 progesterone metabolite allopregnanolone potentiates GABA(A) receptor-mediated inhibition of 5-HT neuronal activity.

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.

Firing of 5-HT neurones in the dorsal and median raphe nucleus in vitro shows differential alpha1-adrenoceptor and 5-HT1A receptor modulation.

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.

GABA(A) receptor modulation of 5-HT neuronal firing in the median raphe nucleus: implications for the action of anxiolytics.

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.

Mechanism for the acute effects of organophosphate pesticides on the adult 5-HT system.

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.

Pharmacogenetic stimulation of cholinergic pedunculopontine neurons reverses motor deficits in a rat model of Parkinson's disease.

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.

Low-level repeated exposure to diazinon and chlorpyrifos decrease anxiety-like behaviour in adult male rats as assessed by marble burying behaviour.

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.

Adult AMPA GLUA1 receptor subunit loss in 5-HT neurons results in a specific anxiety-phenotype with evidence for dysregulation of 5-HT neuronal activity.

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.

Flattening the glucocorticoid rhythm causes changes in hippocampal expression of messenger RNAs coding structural and functional proteins: implications for aging and depression.

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.

Flattening the corticosterone rhythm attenuates 5-HT1A autoreceptor function in the rat: relevance for 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.

In vivo inhibition of neuronal activity in the rat ventromedial prefrontal cortex by midbrain-raphe nuclei: role of 5-HT1A receptors.

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.

GABA receptor modulation of 5-HT neuronal firing: characterization and effect of moderate in vivo variations in glucocorticoid levels.

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.