Boris Mlinar

Sporadic Autonomic Dysregulation and Death Associated with Excessive Serotonin Autoinhibition

Sudden infant death syndrome is the leading cause of death in the postneonatal period in developed countries. Postmortem studies show alterations in serotonin neurons in the brainstem of such infants. However, the mechanism by which altered serotonin homeostasis might cause sudden death is unknown. We investigated the consequences of altering the autoinhibitory capacity of serotonin neurons with the reversible overexpression of serotonin 1A autoreceptors in transgenic mice. Overexpressing mice exhibited sporadic bradycardia and hypothermia that occurred during a limited developmental period and frequently progressed to death. Moreover, overexpressing mice failed to activate autonomic target organs in response to environmental challenges. These findings show that excessive serotonin autoinhibition is a risk factor for catastrophic autonomic dysregulation and provide a mechanism for a role of altered serotonin homeostasis in sudden infant death syndrome.

Suppression of Serotonin Neuron Firing Increases Aggression in Mice

Numerous studies link decreased serotonin metabolites with increased impulsive and aggressive traits. However, although pharmacological depletion of serotonin is associated with increased aggression, interventions aimed at directly decreasing serotonin neuron activity have supported the opposite association. Furthermore, it is not clear if altered serotonin activity during development may contribute to some of the observed associations. Here, we used two pharmacogenetic approaches in transgenic mice to selectively and reversibly reduce the firing of serotonin neurons in behaving animals. Conditional overexpression of the serotonin 1A receptor (Htr1a) in serotonin neurons showed that a chronic reduction in serotonin neuron firing was associated with heightened aggression. Overexpression of Htr1a in adulthood, but not during development, was sufficient to increase aggression. Rapid suppression of serotonin neuron firing by agonist treatment of mice expressing Htr1a exclusively in serotonin neurons also led to increased aggression. These data confirm a role of serotonin activity in setting thresholds for aggressive behavior and support a direct association between low levels of serotonin homeostasis and increased aggression.

Impacts of Brain Serotonin Deficiency following Tph2 Inactivation on Development and Raphe Neuron Serotonergic Specification

Brain serotonin (5-HT) is implicated in a wide range of functions from basic physiological mechanisms to complex behaviors, including neuropsychiatric conditions, as well as in developmental processes. Increasing evidence links 5-HT signaling alterations during development to emotional dysregulation and psychopathology in adult age. To further analyze the importance of brain 5-HT in somatic and brain development and function, and more specifically differentiation and specification of the serotonergic system itself, we generated a mouse model with brain-specific 5-HT deficiency resulting from a genetically driven constitutive inactivation of neuronal tryptophan hydroxylase-2 (Tph2). Tph2 inactivation (Tph2−/−) resulted in brain 5-HT deficiency leading to growth retardation and persistent leanness, whereas a sex- and age-dependent increase in body weight was observed in Tph2+/− mice. The conserved expression pattern of the 5-HT neuron-specific markers (except Tph2 and 5-HT) demonstrates that brain 5-HT synthesis is not a prerequisite for the proliferation, differentiation and survival of raphe neurons subjected to the developmental program of serotonergic specification. Furthermore, although these neurons are unable to synthesize 5-HT from the precursor tryptophan, they still display electrophysiological properties characteristic of 5-HT neurons. Moreover, 5-HT deficiency induces an up-regulation of 5-HT1A and 5-HT1B receptors across brain regions as well as a reduction of norepinephrine concentrations accompanied by a reduced number of noradrenergic neurons. Together, our results characterize developmental, neurochemical, neurobiological and electrophysiological consequences of brain-specific 5-HT deficiency, reveal a dual dose-dependent role of 5-HT in body weight regulation and show that differentiation of serotonergic neuron phenotype is independent from endogenous 5-HT synthesis.

Endogenous 5-HT, released by MDMA through serotonin transporter- and secretory vesicle-dependent mechanisms, reduces hippocampal excitatory synaptic transmission by preferential activation of 5-HT1B receptors located on CA1 pyramidal neurons

A multitude of different serotonin (5‐HT) receptor types are expressed in the hippocampus, but the identity of receptors actually mediating the physiological response to endogenous 5‐HT has not been determined. We combined pharmacologically induced release of 5‐HT with patch‐clamp recordings on disinhibited rat CA1 minislices to determine effects of endogenous 5‐HT on the excitability of pyramidal neurons and synaptic transmission among them. We found that application of 5‐HT releasers, 3,4‐methylenedioxy‐methamphetamine (MDMA) or p‐methylthioamphetamine, at concentrations ranging from 2 to 50 µm, reduced the excitatory synaptic transmission between CA1 pyramidal neurons without altering their basal electrical properties. This effect of MDMA was blocked by the selective 5‐HT1B antagonist GR 55562, was dependent on endogenous 5‐HT content and was mediated by presynaptically located, pertussis‐toxin sensitive mechanisms. We found no other MDMA effects in our preparation, which indicates that the release of endogenous 5‐HT preferentially stimulates 5‐HT1B receptors on CA1 pyramidal neurons. Therefore, 5‐HT1B receptor activation may represent a predominant component of the physiological response to endogenous 5‐HT in the CA1. The high sensitivity of the 5‐HT1B receptor‐mediated reduction of polysynaptic excitatory responses to the extracellular 5‐HT level enabled us to study mechanisms of the 5‐HT releasing action of MDMA. Block of the serotonin transporter (SERT) with citalopram slowed the time course and reduced overall 5‐HT release by MDMA. Depletion of vesicular 5‐HT, by inhibition of vesicular monoamine transporter type 2 with tetrabenazine prevented the release. Thus although the SERT reversal contributes, a direct vesicle‐depleting action is essential for MDMA release of 5‐HT.

Pharmacological characterization of 5‐HT1B receptor‐mediated inhibition of local excitatory synaptic transmission in the CA1 region of rat hippocampus

In the hippocampus, axon collaterals of CA1 pyramidal cells project locally onto neighbouring CA1 pyramidal cells and interneurones, forming a local excitatory network which, in disinhibited conditions, feeds polysynaptic epscs (poly‐epscs). 5‐hydroxytryptamine (5‐HT) has been shown to inhibit poly‐epscs through activation of a presynaptic receptor. The aim of the present work was the pharmacological characterization of the 5‐HT receptor involved in this 5‐HT action. Poly‐epscs, evoked by electrical stimulation of the stratum radiatum and recorded in whole‐cell voltage‐clamp from CA1 pyramidal neurones, were studied in mini‐slices of the CA1 region under pharmacological block of GABAA, GABAB, and 5‐HT1A receptors. The 5‐HT1B receptor selective agonist 1,4‐dihydro‐3‐(1,2,3,6‐tetrahydro‐4‐pyridinyl)‐5H‐pyrrolo[3,2‐b]pyridin‐5‐one dihydrochloride (CP 93129) inhibited poly‐epscs (EC50=55 nM), an effect mimicked by the 5‐HT1B ligands 5‐carboxamidotryptamine (5‐CT; EC50=14 nM) and methylergometrine (EC50=78 nM), but not by 1‐(3‐chlorophenyl)piperazine dihydrochloride (mCPP; 10 μM) or 7‐trifluoromethyl‐4(4‐methyl‐1‐piperazinyl)‐pyrrolo[1,2‐a]quinoxaline dimaleate (CGS 12066B; 10 μM). The effects of CP 93129 and 5‐CT were blocked by the selective 5‐HT1B receptor antagonist 3‐[3‐(dimethylamino)propyl]‐4‐hydroxy‐N‐[4‐(4‐pyridinyl)phenyl]benzamide dihydrochloride (GR 55562; KB∼100 nM) and by cyanopindolol (KB=6 nM); methiothepin (10 μM) and dihydroergotamine (1 μM). For both GR 55562 and methiothepin, application times of at least two hours were required in order to achieve their full antagonistic effects. Our results demonstrate that 5‐HT1B receptors are responsible for the presynaptic inhibition of neurotransmission at CA1/CA1 local excitatory synapses exerted by 5‐HT.

5-HT4 receptor activation induces long-lasting EPSP-spike potentiation in CA1 pyramidal neurons.

Recent studies implicated involvement of the 5‐hydroxytryptamine4 (5‐HT4) receptor in cognitive and emotional processes. The highest 5‐HT4 receptor densities in the brain are found in the limbic system including the hippocampus. Here we used the selective 5‐HT4 receptor full agonist, N‐pentyl‐N′‐aminoguanidine carbazimidamide (SDZ‐216454) to characterize effects of 5‐HT4 receptor activation in whole‐cell and field recordings in the area CA1 in hippocampal slices prepared from 3 to 4‐ and 6 to 9‐week‐old rats, respectively. Extracellular recordings showed that transient 5‐HT4 receptor activation by 10–20 min application of SDZ‐216454 induces field excitatory postsynaptic potential (fEPSP)‐population spike potentiation (ESP5‐HT4), which persisted for as long as we held the recordings (> 2 h). ESP5‐HT4 displayed characteristics different from EPSP‐spike potentiation that accompanies long‐term potentiation; it developed without an associated increase in synaptic transmission, was independent on afferent input, activity of postsynaptic neurons and N‐methyl‐d‐aspartate receptor activation; and was expressed in the presence of GABA receptor antagonists. ESP5‐HT4 was also induced by transient application of the natural neurotransmitter, 5‐HT. The increase in the evoked population spike (PS) induced by SDZ‐216454 was not prevented by blockers of hyperpolarization‐activated cation current (Ih), Cs+ and ZD‐7288, but was mimicked and occluded by 150 µm Ba2+. Whole‐cell voltage‐clamp recordings from pyramidal neurons demonstrated that SDZ‐216454 application increases membrane resistance with a concomitant decrease in a Ba2+‐sensitive inwardly rectifying K+ current and the Ba2+‐insensitive K+ current underlying slow afterhyperpolarization (IsAHP). We conclude that 5‐HT4 receptor activation may cause a long‐lasting excitability increase in CA1 pyramidal neurons by inhibition of a Ba2+‐sensitive inwardly rectifying K+ current.

Conservation of 5-HT1A receptor-mediated autoinhibition of serotonin (5-HT) neurons in mice with altered 5-HT homeostasis

Firing activity of serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) is controlled by inhibitory somatodendritic 5-HT1A autoreceptors. This autoinhibitory mechanism is implicated in the etiology of disorders of emotion regulation, such as anxiety disorders and depression, as well as in the mechanism of antidepressant action. Here, we investigated how persistent alterations in brain 5-HT availability affect autoinhibition in two genetically modified mouse models lacking critical mediators of serotonergic transmission: 5-HT transporter knockout (Sert-/-) and tryptophan hydroxylase-2 knockout (Tph2-/-) mice. The degree of autoinhibition was assessed by loose-seal cell-attached recording in DRN slices. First, application of the 5-HT1A-selective agonist R(+)-8-hydroxy-2-(di-n-propylamino)tetralin showed mild sensitization and marked desensitization of 5-HT1A receptors in Tph2-/- mice and Sert-/- mice, respectively. While 5-HT neurons from Tph2-/- mice did not display autoinhibition in response to L-tryptophan, autoinhibition of these neurons was unaltered in Sert-/- mice despite marked desensitization of their 5-HT1A autoreceptors. When the Tph2-dependent 5-HT synthesis step was bypassed by application of 5-hydroxy-L-tryptophan (5-HTP), neurons from both Tph2-/- and Sert-/- mice decreased their firing rates at significantly lower concentrations of 5-HTP compared to wildtype controls. Our findings demonstrate that, as opposed to the prevalent view, sensitivity of somatodendritic 5-HT1A receptors does not predict the magnitude of 5-HT neuron autoinhibition. Changes in 5-HT1A receptor sensitivity may rather be seen as an adaptive mechanism to keep autoinhibition functioning in response to extremely altered levels of extracellular 5-HT resulting from targeted inactivation of mediators of serotonergic signaling.

Selective inhibition of local excitatory synaptic transmission by serotonin through an unconventional receptor in the CA1 region of rat hippocampus

1 The modulation of synaptic transmission by serotonin (5‐HT) was studied using whole‐cell voltage‐clamp and sharp‐electrode current‐clamp recordings from CA1 pyramidal neurones in transverse rat hippocampal slices in vitro. 2 With GABAA receptors blocked, polysynaptic transmission evoked by stratum radiatum stimulation was inhibited by submicromolar concentrations of 5‐HT, while monosynaptic excitatory transmission and CA1 pyramidal neurone excitability were unaffected. The effect persisted following pharmacological blockade of 5‐HT1A and 5‐HT4 receptors, which directly affect CA1 pyramidal neurone excitability. 3 Concentration‐response relationships for 5‐HT were determined in individual neurones; the EC50 values for block of polysynaptic excitation and inhibition by 5‐HT were ≈230 and ≈160 nm, respectively. The 5‐HT receptor type responsible for the observed effect does not fall easily into the present classification of 5‐HT receptors. 4 5‐HT inhibition of polysynaptic EPSCs persisted following complete block of GABAergic transmission and in CA1 minislices, ruling out indirect effects through interneurones and non‐CA1 pyramidal neurones, respectively. 5 Monosynaptic EPSCs evoked by stimulation of CA1 afferent pathways appeared to be unaffected by 5‐HT. Monosynaptic EPSCs evoked by stimulation of the alveus, which contains CA1 pyramidal neurone axons, were partially inhibited by 5‐HT. 6 We conclude that 5‐HT inhibited synaptic transmission by acting at local recurrent collaterals of CA1 pyramidal neurones. This may represent an important physiological action of 5‐HT in the hippocampus, since it occurs over a lower concentration range than the 5‐HT effects reported so far.

Pharmacological Characterization of 5-HT1A Autoreceptor-Coupled GIRK Channels in Rat Dorsal Raphe 5-HT Neurons

G protein-activated inwardly rectifying potassium (GIRK) channels in 5-HT neurons are assumed to be principal effectors of 5-hydroxytryptamine 1A (5-HT1A) autoreceptors, but their pharmacology, subunit composition and the role in regulation of 5-HT neuron activity have not been fully elucidated. We sought for a pharmacological tool for assessing the functional role of GIRK channels in 5-HT neurons by characterizing the effects of drugs known to block GIRK channels in the submicromolar range of concentrations. Whole-cell voltage-clamp recording in brainstem slices were used to determine concentration-response relationships for the selected GIRK channel blockers on 5-HT1A autoreceptor-activated inwardly rectifying K+ conductance in rat dorsal raphe 5-HT neurons. 5-HT1A autoreceptor-activated GIRK conductance was completely blocked by the nonselective inwardly rectifying potassium channels blocker Ba2+ (EC50 = 9.4 μM, full block with 100 μM) and by SCH23390 (EC50 = 1.95 μM, full block with 30 μM). GIRK-specific blocker tertiapin-Q blocked 5-HT1A autoreceptor-activated GIRK conductance with high potency (EC50 = 33.6 nM), but incompletely, i.e. ~16% of total conductance resulted to be tertiapin-Q-resistant. U73343 and SCH28080, reported to block GIRK channels with submicromolar EC50s, were essentially ineffective in 5-HT neurons. Our data show that inwardly rectifying K+ channels coupled to 5-HT1A autoreceptors display pharmacological properties generally expected for neuronal GIRK channels, but different from GIRK1-GIRK2 heteromers, the predominant form of brain GIRK channels. Distinct pharmacological properties of GIRK channels in 5-HT neurons should be explored for the development of new therapeutic agents for mood disorders.

Enhanced hippocampal long-term potentiation following repeated MDMA treatment in Dark-Agouti rats

In rats and primates, (±)3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) produces both long-lasting damage to serotonergic axons and memory impairment. Our objective was to determine effects of neurotoxic dose of MDMA on long-term potentiation (LTP) in hippocampal area CA1 in Dark-Agouti (DA) rats. One week after neurotoxic MDMA treatment in vivo (12.5mg/kg i.p., once a week, per three weeks), serotonergic deficit was evident in hippocampal slices as 56.3% reduction in 5-HT content (p=0.04) and as 68.4% reduction in the effect of endogenous 5-HT release on synaptic neurotransmission (p<0.01). In hippocampal slices from the same animals, LTP was on average 46% greater than that observed in sham-treated controls (42.9 ± 3.5%; n=12 vs. 29.2 ± 3.2%; n=12; p<0.01). Non-neurotoxic dose of MDMA (12.5 mg/kg, i.p., one time) did not change LTP one week after the treatment, suggesting correlation between serotonergic deficit and enhanced synaptic plasticity. We conclude that MDMA-induced impairment of learning and memory is not a consequence of hippocampal LTP inhibition.