Neurophysiological signals as potential translatable biomarkers for modulation of metabotropic glutamate 5 receptors.

The Group I metabotropic glutamate receptor subtype 5 (mGluR5) is widely distributed in the brain with dense expression in the cerebral cortex, hippocampus, and basal ganglia. These receptors have been implicated in psychiatric and neurological disorders such as schizophrenia, Fragile X syndrome, addiction, anxiety/depression, Parkinson's disease and neuropathic pain. The present study evaluated the effects of the mGluR5 negative allosteric modulators (NAMs) 4-difluoromethoxy-3-(pyridine-2-ylethynyl)phenyl)5H-pyrrolo[3,4-b]pyridine-6(7H)-yl methanone (GRN-529) and methyl (3aR,4S,7aR)-4-hydroxy-4-[(3-methylphenyl)ethynyl]octahydro-1H-indole-1-carboxylate (AFQ056) on polysomnographic (PSG) and quantitative electroencephalographic (qEEG) measures in freely moving rats. Furthermore, the anxiolytic profile of GRN-529 was characterized in anesthetized rats by measuring stimulation-induced hippocampal theta oscillation. The present findings demonstrate that inhibition of mGluR5 via its allosteric site profoundly modulates high-level neuronal network activities as indicated by changes in sleep-wake activity and power distribution of qEEG. Both GRN-529 and AFQ056 reduced the total time spent in rapid-eye movement with AFQ056 producing a significant increase in wakefulness at the highest dose tested. Additionally, qEEG revealed significant compound-induced increases in delta power concomitant with more subtle decreases in theta and alpha band power. Receptor occupancy (RO) studies revealed that GRN-529 and AFQ056 at all doses resulted in over 45% mGluR5 occupancy. Furthermore, GRN-529 dose-dependently decreased elicited hippocampal theta frequency, consistent with previous findings using clinically active anxiolytic compounds. The described changes in neurophysiological signals identified in freely moving rats may be considered suitable translational biomarkers for the clinical evaluation of mGluR5 NAMs.

Rhythmic theta and delta activity of cortical and hippocampal neuronal networks in genetically or pharmacologically induced N-methyl-D-aspartate receptor hypofunction under urethane anesthesia.

N-Methyl-d-aspartate receptor (NMDAR) antagonists mimic several symptoms of schizophrenia in healthy subjects, and are used in preclinical disease models. In the present study, the impact of pharmacologically and genetically induced NMDAR hypofunction was assessed in rats and mice, including the NMDAR hypomorphic (Grin1) mice, with respect to neuronal network oscillations. Field potentials were recorded from the ventro-medial prefrontal cortex (mPFC) and hippocampus (CA1) in rats, as well as spontaneous and elicited hippocampal theta oscillations in response to brainstem stimulation in Grin1 and wild-type (WT) mice under anesthesia. Effects of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor positive allosteric modulator LY451395 were tested in Grin1 mice and in WT mice following an MK-801 challenge. Recordings from the mPFC and CA1 in rats revealed regular delta and theta oscillations, respectively, which were disrupted by MK-801. In WT mice, MK-801 reduced both spontaneous and elicited hippocampal theta power. Age-matched Grin1 mice showed abnormal hippocampal field potentials, resembling activity seen after administration of MK-801 in WT mice, but also epileptiform discharges. Administration of MK-801 achieved high levels of NMDAR occupancy (84-98%) in both rats and mice, which is comparable to the approximately 90-95% reduction of NMDAR expression in the Grin1 mouse. Impaired elicited CA1 theta oscillation in WT mice following MK-801, or Grin1 mice was significantly improved by LY451395. These findings demonstrate similar, although not identical, changes in network activity following reduction in functioning NMDARs induced by acute pharmacological or genetic manipulations, indicating that these novel neurophysiological models could be used in evaluating drug candidates targeting glutamate neurotransmission.

Nicotine decreases DNA methyltransferase 1 expression and glutamic acid decarboxylase 67 promoter methylation in GABAergic interneurons.

Tobacco smoking is frequently abused by schizophrenia patients (SZP). The major synaptically active component inhaled from cigarettes is nicotine, hence the smoking habit of SZP may represent an attempt to use nicotine self-medication to correct (i) a central nervous system nicotinic acetylcholine receptor (nAChR) dysfunction, (ii) DNA-methyltransferase 1 (DMT1) overexpression in GABAergic neurons, and (iii) the down-regulation of reelin and GAD(67) expression caused by the increase of DNMT1-mediated hypermethylation of promoters in GABAergic interneurons of the telencephalon. Nicotine (4.5-22 micromol/kg s.c., 4 injections during the 12-h light cycle for 4 days) decreases DNMT1 mRNA and protein and increases GAD(67) expression in the mouse frontal cortex (FC). This nicotine-induced decrease of DNMT1 mRNA expression is greater (80%) in laser microdissected FC layer I GABAergic neurons than in the whole FC (40%), suggesting selectivity differences for the specific nicotinic receptor populations expressed in GABAergic neurons of different cortical layers. The down-regulation of DNMT1 expression induced by nicotine in the FC is also observed in the hippocampus but not in striatal GABAergic neurons. Furthermore, these data show that in the FC, the same doses of nicotine that decrease DNMT1 expression also (i) diminished the level of cytosine-5-methylation in the GAD(67) promoter and (ii) prevented the methionine-induced hypermethylation of the same promoter. Pretreatment with mecamylamine (6 micromol/kg s.c.), an nAChR blocker that penetrates the blood-brain barrier, prevents the nicotine-induced decrease of FC DNMT1 expression. Taken together, these results suggest that nicotine, by activating nAChRs located on cortical or hippocampal GABAergic interneurons, can up-regulate GAD(67) expression via an epigenetic mechanism. Nicotine is not effective in striatal medium spiny GABAergic neurons that primarily express muscarinic receptors.

Preclinical characterization of selective phosphodiesterase 10A inhibitors: a new therapeutic approach to the treatment of schizophrenia.

We have recently proposed the hypothesis that inhibition of the cyclic nucleotide phosphodiesterase (PDE) 10A may represent a new pharmacological approach to the treatment of schizophrenia (Curr Opin Invest Drug 8:54-59, 2007). PDE10A is highly expressed in the medium spiny neurons of the mammalian striatum (Brain Res 985:113-126, 2003; J Histochem Cytochem 54:1205-1213, 2006; Neuroscience 139:597-607, 2006), where the enzyme is hypothesized to regulate both cAMP and cGMP signaling cascades to impact early signal processing in the corticostriatothalamic circuit (Neuropharmacology 51:374-385, 2006; Neuropharmacology 51:386-396, 2006). Our current understanding of the physiological role of PDE10A and the therapeutic utility of PDE10A inhibitors derives in part from studies with papaverine, the only pharmacological tool for this target extensively profiled to date. However, this agent has significant limitations in this regard, namely, relatively poor potency and selectivity and a very short exposure half-life after systemic administration. In the present report, we describe the discovery of a new class of PDE10A inhibitors exemplified by TP-10 (2-{4-[-pyridin-4-yl-1-(2,2,2-trifluoro-ethyl)-1H-pyrazol-3-yl]-phenoxymethyl}-quinoline succinic acid), an agent with greatly improved potency, selectivity, and pharmaceutical properties. These new pharmacological tools enabled studies that provide further evidence that inhibition of PDE10A represents an important new target for the treatment of schizophrenia and related disorders of basal ganglia function.

Modulation of hippocampal theta oscillation by histamine H3 receptors.

Preclinical findings demonstrate procognitive actions of histamine 3 (H3) receptor antagonists/inverse agonists. Since a prominent role of neuronal network oscillations of the hippocampus, such as theta band oscillation, has been recognized in numerous cognitive functions, in the present study, the potential involvement of H3 receptors in modulation of hippocampal theta activity has been investigated using various recording paradigms. Systemic administration of the selective H3 receptor antagonists/inverse agonists, thioperamide and ciproxifan (0.1 mg/kg to 1 mg/kg i.v.), dose dependently increased hippocampal theta power, similarly to methylphenidate (0.1-1 mg/kg i.v.), in chloral hydrate anesthetized rats. When hippocampal theta oscillation was elicited by electrical brainstem (nucleus pontis oralis) stimulation, ciproxifan (1 mg/kg i.v.) augmented the power of stimulation-induced theta. In contrast, systemic administration of methylphenidate (1 mg/kg i.v.) did not modify elicited theta. To analyze the role of H3 receptors on stage- and behavior-dependent hippocampal theta activity, polysomnographic recordings were carried out together with field potential recordings at the hippocampal fissure in freely moving rats for 8 h during the light phase of the circadian cycle. Systemic administration of ciproxifan (3.0 mg/kg, i.p.) promoted wakefulness with a concomitant reduction in cortical delta power and augmented novelty-induced hippocampal theta activity. These findings provide evidence that H3 receptors play an important role in regulation of hippocampal theta oscillation, representing one of the probable mechanisms involved in histamine-induced modulation of higher brain functions, such as attention and learning.

Activation of alpha7 acetylcholine receptors augments stimulation-induced hippocampal theta oscillation.

In the septohippocampal formation alpha7 nicotinic receptors (alpha7 nAChRs) are predominantly expressed by neurons well positioned to modulate hippocampal theta oscillation, such as GABAergic interneurons in the hippocampus, and by both GABAergic and cholinergic septal neurons. In the present experiments, we evaluated the efficacy of the recently developed selective alpha7 nAChR agonist PNU-282987 on hippocampal theta oscillation in anaesthetized rats. This compound shows high affinity for the rat alpha7 nAChRs (Ki = 26 nM) but a negligible activity at other nAChRs. Systemic administration of PNU-282987 significantly enhanced the power (by 40%) of hippocampal theta oscillation induced by electrical stimulation of the brainstem reticular formation. In contrast, the amnesic and muscarinic receptor antagonist scopolamine significantly decreased the power (by 68%) of the stimulation-induced theta oscillation. Given the connection between hippocampal theta oscillation and cognitive processes, it is proposed that precognitive actions of alpha7 nAChR agonists could be mediated, at least in part, by modulation of hippocampal oscillatory activity.

The selective alpha7 nicotinic acetylcholine receptor agonist PNU-282987 [N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride] enhances GABAergic synaptic activity in brain slices and restores auditory gating deficits in anesthetized rats.

Schizophrenic patients are thought to have an impaired ability to process sensory information. This deficit leads to disrupted auditory gating measured electrophysiologically as a reduced suppression of the second of paired auditoryevoked responses (P50) and is proposed to be associated with decreased function and/or expression of the homomeric alpha7 nicotinic acetylcholine receptor (nAChR). Here, we provide evidence that N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987), a novel selective agonist of the alpha7 nAChR, evoked whole-cell currents from cultured rat hippocampal neurons that were sensitive to the selective alpha7 nAChR antagonist methyllycaconitine (MLA) and enhanced GABAergic synaptic activity when applied to hippocampal slices. Amphetamine-induced sensory gating deficit, determined by auditory-evoked potentials in hippocampal CA3 region, was restored by systemic administration of PNU-282987 in chloral hydrate-anesthetized rats. Auditory gating of rat reticular thalamic neurons was also disrupted by amphetamine; however, PNU-282987 normalized gating deficit only in a subset of tested neurons (6 of 11). Furthermore, PNU-282987 improved the inherent hippocampal gating deficit occurring in a subpopulation of anesthetized rats, and enhanced amphetamine-induced hippocampal oscillation. We propose that the alpha7 nAChR agonist PNU-282987, via modulating/enhancing hippocampal GABAergic neurotransmission, improves auditory gating and enhances hippocampal oscillatory activity. These results provide further support for the concept that drugs that selectively activate alpha7 nAChRs may offer a novel, potential pharmacotherapy in treatment of schizophrenia.

Modulation of septo-hippocampal Theta activity by GABAA receptors: an experimental and computational approach.

Theta frequency oscillation of the septo-hippocampal system has been considered as a prominent activity associated with cognitive function and affective processes. It is well documented that anxiolytic drugs diminish septo-hippocampal oscillatory Theta activity contributing to their either therapeutic or unwanted side effects. In the present experiments we applied a combination of computational and physiological techniques to explore the functional role of GABAA receptors in Theta oscillation. In electrophysiological experiments extracellular single unit recordings were performed from medial septum/diagonal band of Broca with simultaneous hippocampal (CA1) electroencephalogram (EEG) recordings from anesthetized rats. Neurotransmission at GABAA receptors were modulated by means of pharmacological tools: the actions of the GABAA receptor positive allosteric modulator diazepam and inverse agonist/negative allosteric modulator FG-7142 were evaluated on septo-hippocampal activity. Systemic administration of diazepam inhibited, whereas FG-7142 enhanced Theta oscillation of septal neurons and hippocampal EEG Theta activity. In parallel to these experimental observations, a computational model has been constructed by implementing a septal GABA neuron model with a CA1 hippocampal model containing three types of neurons (including oriens and basket interneurons and pyramidal cells; latter modeled by multicompartmental techniques; for detailed model description with network parameters see online addendum: http://geza.kzoo.edu/theta). This connectivity made the network capable of simulating the responses of the septo-hippocampal circuitry to the modulation of GABAA transmission, and the presently described computational model proved suitable to reveal several aspects of pharmacological modulation of GABAA receptors. In addition, computational findings indicated different roles of distinctively located GABAA receptors in theta generation.

Evidence for a role of GABA interneurones in the cortical modulation of midbrain 5-hydroxytryptamine neurones.

Recent electrophysiological studies demonstrate that the ventral medial prefrontal cortex has a powerful inhibitory influence on 5-hydroxytryptamine (5-HT) neurones in the dorsal raphe nucleus. Here we utilised a combination of anatomical and electrophysiological methods to characterise the cellular substrate underlying this effect.Anterograde tracing (Phaseolus vulgaris leucoagglutinin) using electron microscopy demonstrated a pathway from the ventral medial prefrontal cortex that makes neuronal contacts throughout the dorsal raphe nucleus. These contacts were predominantly asymmetrical synapses adjoining GABA immunoreactive dendrites and spines. In vivo extracellular recordings were made in the dorsal raphe nucleus of the anaesthetised rat from a subpopulation of non-5-HT neurones. These neurones were fast-firing, irregular and with short spike width, properties strongly reminiscent of immunochemically identified GABA interneurones in other brain regions. Recordings of classical 5-HT neurones were also included. Electrical stimulation of the ventral medial prefrontal cortex elicited a rapid onset (16 ms latency), orthodromic excitation of the non-5-HT neurones (13/25 neurones). This stimulation also caused a pronounced inhibition of most 5-HT neurones tested, with a longer latency (30 ms), and this was partially blocked by locally applied bicuculline. These data provide the first evidence that the ventral medial prefrontal cortex influences the activity of large numbers of raphe 5-HT neurones by targeting a local network of GABA neurones. This circuitry predicts that physiological and pathological changes in the ventral medial prefrontal cortex will impact on significant parts of the forebrain 5-HT system.

Different tonic regulation of neuronal activity in the rat dorsal raphe and medial prefrontal cortex via 5-HT(1A) receptors.

It has been established that 5-HT(1A) receptors are expressed both presynaptically as autoreceptors by 5-HT containing neurones, and postsynaptically by a variety of other neurones. Activation of either somatodendritic 5-HT(1A) autoreceptors or postsynaptic 5-HT(1A) receptors induces hyperpolarisation and inhibition of action potential discharge of the neurones, but it is unclear whether 5-HT(1A) receptors are under a general tonic influence by 5-HT. In the present study, using single unit recordings from both anesthetized and non-anesthetized rats, we show that the activity of neurones in the medial prefrontal cortex is not altered by systemic administration of the selective 5-HT(1A) receptor antagonist, WAY 100635. In contrast, WAY 100635 increased the firing rate of 5-HT neurones in the dorsal raphe nucleus. Our findings indicate a tonic activation of presynaptic somatodendritic but not postsynaptic cortical 5-HT(1A) receptors.

Nicotine inhibits firing activity of dorsal raphe 5-HT neurones in vivo.

It is established that the brain monoaminergic systems are among the main targets of several dependence-inducing drugs, including nicotine. In the present study extracellular electrophysiological recordings were performed to investigate the effects of nicotine on dorsal raphe 5-HT neurones. Nicotine, administered systemically (50-400 microg/kg, i.v.) in chloral hydrate-anaesthetised rats, induced a transient inhibition of the majority of 5-HT neurones recorded (38 of 45). The inhibition was rapid in onset (about 30 s) and the firing rate returned to baseline within 1-3 min. No apparent tachyphylaxis was observed to this inhibitory effect. The centrally acting nicotine antagonist mecamylamine (4 mg/kg, i.v.), but not the peripherally acting nicotine antagonist chlorisondamine (0.3 mg/kg, i.v.) antagonised the nicotine-induced inhibition of 5-HT neurones. The inhibition of 5-HT neurones was also blocked with a selective 5-HT1A receptor antagonist (WAY 100635; 0.1 mg/kg, i.v.), indicating a possible involvement of somato-dendritic 5-HT1A receptors in the effect of nicotine. Interestingly, microiontophoretic application of nicotine into the dorsal raphe failed to inhibit 5-HT neurones, suggesting an indirect effect of nicotine on 5-HT neurones, possibly involving afferent pathways.

Nicotine-induced excitation of locus coeruleus neurons is blocked by elevated levels of endogenous kynurenic acid.

The present electrophysiological study shows that manipulation with endogenous brain kynurenic acid (KYNA) is able to affect the response of central noradrenergic neurons to nicotine. Previous studies have shown that systemically administered nicotine in low doses is associated with a marked, but short-lasting increase in the firing rate of rat noradrenergic neurons in the locus coeruleus (LC). This action of nicotine is of peripheral origin and finally mediated via a release of glutamate within the LC. KYNA is an endogenous glutamate receptor antagonist, which shows an uneven distribution in human brain. Previous studies have shown that a potent inhibitor of kynurenine 3-hydroxylase, PNU 156561A, is able to dose-dependently increase the levels of KYNA in brain. Anesthetized rats were given PNU 156561A in a dose that caused a 5-fold increase in brain KYNA levels after 3-6 hours (40 mg/kg, i.v. ). This treatment was found to abolish the increase in firing rate of LC neurons induced by nicotine (25-200 microg/kg, i.v.). The results of the present study show that an increased concentration of endogenous brain KYNA is able to inhibit the activation of central noradrenergic neurons by nicotine. In addition, our results highlight the role of endogenous KYNA in brain as a potentially important modulator of brain glutamatergic responses.

Neurochemical and electrophysiological studies on the functional significance of burst firing in serotonergic neurons.

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.

Brain uncoupling protein 2: uncoupled neuronal mitochondria predict thermal synapses in homeostatic centers.

Distinct brain peptidergic circuits govern peripheral energy homeostasis and related behavior. Here we report that mitochondrial uncoupling protein 2 (UCP2) is expressed discretely in neurons involved in homeostatic regulation. UCP2 protein was associated with the mitochondria of neurons, predominantly in axons and axon terminals. UCP2-producing neurons were found to be the targets of peripheral hormones, including leptin and gonadal steroids, and the presence of UCP2 protein in axonal processes predicted increased local brain mitochondrial uncoupling activity and heat production. In the hypothalamus, perikarya producing corticotropin-releasing factor, vasopressin, oxytocin, and neuropeptide Y also expressed UCP2. Furthermore, axon terminals containing UCP2 innervated diverse hypothalamic neuronal populations. These cells included those producing orexin, melanin-concentrating hormone, and luteinizing hormone-releasing hormone. When c-fos-expressing cells were analyzed in the basal brain after either fasting or cold exposure, it was found that all activated neurons received a robust UCP2 input on their perikarya and proximal dendrites. Thus, our data suggest the novel concept that heat produced by axonal UCP2 modulates neurotransmission in homeostatic centers, thereby coordinating the activity of those brain circuits that regulate daily energy balance and related autonomic and endocrine processes.

Role of the medial prefrontal cortex in 5-HT1A receptor-induced inhibition of 5-HT neuronal activity in the rat.

1. We examined the involvement of the frontal cortex in the 5-HT2A receptor-induced inhibition of 5-HT neurones in the dorsal raphe nucleus (DRN) of the anaesthetized rat using single-unit recordings complemented by Fos-immunocytochemistry. 2. Both transection of the frontal cortex as well as ablation of the medial region of the prefrontal cortex (mPFC) significantly attenuated the inhibition of 5-HT neurones induced by systemic administration of the 5-HT1A receptor agonist, 8-OH-DPAT (0.5-16 microg kg(-1), i.v.). In comparison, the response to 8-OH-DPAT was not altered by ablation of the parietal cortex. The inhibitory effect of 8-OH-DPAT was reversed by the 5-HT1A receptor antagonist, WAY 100635 (0.1 mg kg(-1), i.v.) in all neurones tested. 3. In contrast, cortical transection did not alter the sensitivity of 5-HT neurones to iontophoretic application of 8-OH-DPAT into the DRN. Similarly, cortical transection did not alter the sensitivity of 5-HT neurones to systemic administration of the selective 5-HT reuptake inhibitor, paroxetine (0.1-0.8 mg kg(-1) , i.v.). 4. 8-OH-DPAT evoked excitation of mPFC neurones at doses (0.5-32 microg kg(-1), i.v.) in the range of those which inhibited 5-HT cell firing. At higher doses (32-512 microg kg(-1), i.v.) 8-OH-DPAT inhibited mPFC neurones. 8-OH-DPAT (0.1 mg kg(-1), s.c.) also induced Fos expression in the mPFC. The neuronal excitation and inhibition, as well as the Fos expression, were antagonized by WAY 100635. 5. These data add further support to the view that the inhibitory effect of 5-HT1A receptor agonists on the firing activity of DRN 5-HT neurones involves, in part, activation of a 5-HT1A receptor-mediated postsynaptic feedback loop centred on the mPFC.

An electrophysiological and neuroanatomical study of the medial prefrontal cortical projection to the midbrain raphe nuclei in the rat.

In this study we utilized electrophysiological and pathway tracing methods to investigate the projections from the medial prefrontal cortex to the midbrain raphe nuclei of the rat. Initial pathway tracing experiments using retrograde (horseradish peroxidase conjugates with wheatgerm agglutinin or choleratoxin B subunit) and anterograde (Phaseolus vulgaris-leucoagglutinin) markers demonstrated a direct, bilateral projection to the dorsal raphe nucleus and median raphe nucleus from the medial prefrontal cortex, and the origin of this projection was localized predominantly in the ventral medial prefrontal cortex (infralimbic/dorsal penduncular cortices). Using chloral hydrate-anaesthetized rats, extracellular recordings were made mostly from 5-hydroxytryptamine neurons in the dorsal raphe nucleus, but non-5-hydroxytryptamine dorsal raphe neurons were also studied, as was a small number of 5-hydroxytryptamine neurons in the median raphe nucleus. In an initial study, electrical stimulation of the ventral medial prefrontal cortex caused a post-stimulus inhibition in the majority (49/56) of dorsal raphe 5-hydroxytryptamine neurons tested (mean duration of inhibition, 200+/-17 ms); in some cases (8/56) the inhibition was preceded by short-latency (26 +/-3 ms) orthodromic activation, and a small number of cells was antidromically activated (6/56). Both single spiking and burst-firing 5-hydroxytryptamine neurons in the dorsal raphe nucleus responded in the same way, and median raphe 5-hydroxytryptamine neurons were also inhibited (5/5). In contrast, few (2/12) of the non-5-hydroxytryptamine dorsal raphe neurons tested were inhibited by ventral medial prefrontal cortex stimulation. The effects of stimulation of the dorsal and ventral medial prefrontal cortex were compared on the same raphe 5-hydroxytryptamine neurons (n=17): ventral medial prefrontal cortex stimulation inhibited 16/17 of these neurons while only 8/17 were inhibited by dorsal medial prefrontal cortex stimulation. Finally, the inhibitory effect of ventral medial prefrontal cortex stimulation on 5-hydroxytryptamine cell-firing was not altered by 5-hydroxytryptamine depletion with p-chlorophenylalanine or by systemic administration of the selective 5-hydroxytryptamine1A receptor antagonist WAY 100635. The latter findings indicate that the inhibition is not due to release of raphe 5-hydroxytryptamine which could theoretically arise from anti- or orthodromically activated 5-hydroxytryptamine neurons. Our results show that stimulation of the ventral medial prefrontal cortex causes a marked post-stimulus inhibition in the vast majority of midbrain raphe 5-hydroxytryptamine neurons tested. It seems likely that the projection from ventral medial prefrontal cortex to the midbrain raphe nuclei mediates the responses of 5-hydroxytryptamine neurons to cortical stimulation. These data are relevant to recent discoveries of functional and structural abnormalities in the medial prefrontal cortex of patients with major depressive illness.

Electrophysiological and neurochemical evidence that pindolol has agonist properties at the 5-HT1A autoreceptor in vivo.

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.

Burst-firing activity of presumed 5-HT neurones of the rat dorsal raphe nucleus: electrophysiological analysis by antidromic stimulation.

We recently reported raphe neurones which frequently fired spikes in short bursts. However, the action potentials were broad and the neurones fired in a slow and regular pattern, suggesting they were an unusual type of 5-hydroxytryptamine (5-HT) neurone. In the present study, we investigated whether these putative burst-firing 5-HT neurones project to the forebrain and whether all spikes fired in bursts propagate along the axon. In anaesthetised rats, electrical stimulation of the medial forebrain bundle evoked antidromic spikes in both burst-firing neurones and in single-spiking, classical 5-HT neurones recorded in the dorsal raphe nucleus. Although the antidromic spike latency of the single-spiking and burst-firing neurones showed a clear overlap, burst-firing neurones had a significantly shorter latency than single-spiking neurones. For both burst-firing neurones and classical 5-HT neurones, antidromic spikes made collisions with spontaneously occurring spikes. Furthermore, in all burst-firing neurones tested, first, second and third order spikes in a burst could be made to collide with antidromic spike. Interestingly, in a small number of burst-firing neurones, antidromic stimulation evoked spike doublets, similar to those recorded spontaneously. From these data we conclude that burst-firing neurones in the dorsal raphe nucleus project to the forebrain, and each spike generated by the burst propagates along the axon and could thereby release transmitter (5-HT).

Intracellular recordings from burst-firing presumed serotonergic neurones in the rat dorsal raphe nucleus in vivo.

Here we report the existence of burst-firing neurones in the rat dorsal raphe as detected in vivo using intracellular electrophysiological techniques. These neurones discharged single action potentials and doublets or triplets of action potentials in a slow and regular pattern. The apparent input resistance, action potential width and firing threshold of these burst-firing raphe neurones were indistinguishable from classical 5-HT neurones. Spike doublets were evoked by depolarising DC currents, but only in burst-firing neurones. These findings provide further evidence to support the hypothesis that 5-HT neurones (or a sub-set of them) are capable of burst-firing activity.