Heterogeneous distribution of the serotonin 5‐HT1A receptor mRNA in chemically identified neurons of the mouse rostral brainstem: Implications for the role of serotonin in the regulation of wakefulness and REM sleep

The 5‐HT_1A receptor (5‐HT_1AR) plays a key role in the inhibitory influence of serotonin (5‐HT) on rapid eye movement (REM) sleep in rodents. However, the neuronal networks mediating such influence are mostly unknown, notably in the mouse. This led us to map 5‐HT_1AR mRNA, by in situ hybridization histochemistry (ISHH), and to characterize the neuronal phenotype of 5‐HT_1AR mRNA‐positive neurons by dual ISHH and ISHH combined with immunohistochemistry, throughout the mouse rostral brainstem, a pivotal region for the generation of REM sleep and cortical activation. 5‐HT_1AR mRNA was found in most 5‐HT neurons in the dorsal raphe (DR), the median raphe (MnR), the B9, and the interpeduncular (IP) nuclei. 5‐HT_1AR mRNA‐positive neurons were also identified in individualized clusters of γ‐aminobutyric acid (GABA)ergic neurons in the DR and in neurons of an undetermined phenotype in the MnR. In addition, 1) GABAergic neurons of the ventral portion of Gudden's dorsal tegmental nucleus (DTg), the IP, and the caudal portion of the deep mesencephalic nucleus (DpMe), and 2) glutamatergic neurons scattered in the caudal pontine reticular nucleus (PnC) and densely packed in the internal lateral parabrachial subnucleus (PBil) also expressed 5‐HT_1AR mRNA. In contrast, no specific 5‐HT_1AR‐related ISHH signal was generally detected in brainstem cholinergic and catecholaminergic neurons. These results emphasize the role of 5‐HT_1AR as an autoreceptor and the phenotypical heterogeneity of 5‐HT_1AR‐expressing neurons within the DR and the MnR in the mouse brain. They also provide a neuroanatomical basis for understanding the influence of 5‐HT_1AR on REM sleep and wakefulness. J. Comp. Neurol. 518:2744–2770, 2010. © 2010 Wiley‐Liss, Inc.     

Decreased cerebral blood flow in the limbic and prefrontal cortex using SPECT imaging in a cohort of completed suicides

Suicide has a high comorbidity with impulsivity and depression, and finding imaging biomarkers indicative of patients at high risk for suicidal behavior is invaluable to the clinician. Using single-photon emission computed tomography (SPECT) imaging, we have previously reported regional cerebral blood flow (rCBF) decreases in the medial prefrontal cortex, ventral tegmental area and subgenual cingulate cortex (Brodmann area 25 (BA 25)), a region found to be hypoperfused with treatment-resistant depression. From 2007 to 2010, we have extended our analysis to include nine additional completed suicides. In all, 27 healthy, age- and gender-matched subjects from a previously acquired healthy brain study served as controls to our 21 completed suicides. All 21 suicides had been previously diagnosed with depression according to Diagnostic and Statistical Manual of Mental Disorder-IV criterion. Voxel-by-voxel analyses were performed using statistical parametric mapping to compare the differences in technetium-99m hexamethylpropylene amine oxime brain uptake between the groups. Factor analysis of the data identified the top 10 regions of hypoperfusion in the suicidal group, including the bilateral superior frontal lobes, the right precuneus, the rolandic operculum, postcentral gyrus, left caudate and insular cortex. We also demonstrate more focal decreases in rCBF in the subgenual cingulate cortex (BA 25) in 18 subjects, supporting our previous hypothesis that hypoperfusion of BA 25 may be a risk factor for suicide in depressed patients. This work suggests that SPECT might be useful in predicting risk for suicide completion in subjects with depression or treatment-resistant depression. Further investigation of this work is necessary to better understand the predictive value of this finding.     

High‐frequency stimulation of the medial prefrontal cortex decreases cellular firing in the dorsal raphe

High‐frequency deep brain stimulation (HFS‐DBS) of the subcallosal cingulate (SCC) region has been investigated as a treatment for refractory forms of depression with a ~50% remission rate in open label studies. However, the therapeutic mechanisms of DBS are still largely unknown. Using anaesthetized Sprague Dawley rats, we recorded neuronal spiking activity in 102 neurons of the dorsal raphe (DR) before, during and after the induction of a 5‐min HFS train in the infralimbic region (IL) of the medial prefrontal cortex (mPFC), the rodent homologue of the human SCC. The majority of DR cells (82%) significantly decreased firing rate during HFS (P < 0.01, 55.7 ± 4.5% of baseline, 35 rats). To assess whether mPFC‐HFS mediates inhibition of DR cellular firing by stimulating local GABAergic interneurons, the GABA_A antagonist bicuculline (Bic, 100 μm ) was injected directly into the DR during HFS. Neurons inhibited by HFS recovered their firing rate during Bic+HFS (P < 0.01, n = 15, seven rats) to levels not different from baseline. Cells that were not affected by HFS did not change firing rate during Bic+HFS (P = 0.968, n = 7, three rats). These results indicate that blocking GABA_A reverses HFS‐mediated inhibition of DR neurons. As the cells that were not inhibited by HFS were also unaffected by HFS+Bic, they are probably not innervated by local GABA. Taken together, our results suggest that mPFC‐HFS may exert a preferential effect on DR neurons with GABA_A receptors.     

5-HT6 receptor antagonist SB-271046 enhances extracellular levels of monoamines in the rat medial prefrontal cortex

The present study investigated the neurochemical effects of the selective 5-HT(6) receptor antagonist SB-271046 in the rat medial prefrontal cortex (mPFC). The effect of SB-271046 on extracellular levels of dopamine (DA), norepinephrine (NE), and serotonin (5-HT) in the mPFC was examined using in vivo microdialysis in the freely moving rat. SB-271046 (10 mg/kg, p.o.) produced a significant increase in extracellular levels of both DA and NE without altering 5-HT neurotransmission. These results further support the rationale for the use of 5-HT(6) receptor antagonists in the treatment of cognitive dysfunction associated with psychiatric diseases.     

CSF 5-HIAA, suicide intent and hopelessness in the prediction of early suicide in male high-risk suicide attempters

OBJECTIVE: To study the predictive value of the Beck Suicide Intent Scale (SIS), the Beck Hopelessness Scale (BHS) and of 5-hydroxyindoleacetic acid (5-HIAA) in the cerebrospinal fluid for future early suicide in a group of high-risk male suicide attempters. METHOD: Fifteen consecutive male suicide attempters admitted to a psychiatric ward at the Karolinska Hospital, who were not receiving any treatment with antidepressants were diagnosed according to DSM-III, assessed with SIS and BHS and submitted to lumbar puncture. All patients were followed up for cause of death. RESULTS: Five early suicides (within 2 years) were identified. Mean cerebrospinal fluid (CSF) 5-HIAA differed between suicides and survivors. Low CSF 5-HIAA was identified in those who committed early suicide. Neither the Suicide Intent Score nor the Hopelessness Score distinguished suicides from survivors. CONCLUSION: In high suicide risk hospitalized male psychiatric patients CSF 5-HIAA may be a better predictor of early suicide after attempted suicide than SIS or BHS.     

Serotonin‐transporter‐linked promoter region polymorphism and serotonin transporter binding in drug‐naïve patients with major depression

Aims: Both the serotonin transporter and its genetic regulation by the serotonin‐transporter‐linked polymorphic region have a role in the pathophysiology of depression. Most of the previous studies have found no influence of serotonin‐transporter‐linked polymorphic region allelic variation on serotonin transporter binding in healthy controls or patients with major depression. Due to the inconsistency of the previous findings, we compared single photon emission computed tomography imaging with the serotonin‐transporter‐linked polymorphic region genotype in patients with major depressive disorder. Methods: A total of 23 drug‐naïve patients with major depressive disorder were genotyped and brain imaged with ^[123I]nor‐β‐CIT single photon emission computed tomography. The severity of depression was evaluated with the 17‐item Hamilton depression rating scale. Results: Depressed patients homozygous for the short allele had lower ^[123I]nor‐β‐CIT binding in the medial prefrontal cortex, but not in the midbrain, compared with the other genotypes. Conclusion: The decreased medial prefrontal cortical serotonin transporter binding in the patients homozygous for the short allele may be linked to altered function of the serotonin‐transporter‐linked polymorphic region gene expressed in these patients, especially in the medial prefrontal cortex.     

Elevated synaptophysin I in the prefrontal cortex of human chronic alcoholics

Convergent lines of evidence suggest potentiation of glutamatergic synapses after chronic ethanol exposure, and indicate that the presynaptic effect hereof is on modulators of synaptic strength rather than on executors of glutamate release. To address this hypothesis in the context of ethanol dependence in humans, we used semiquantitative immunoblotting to compare the immunoreactivities of synaptophysin I, syntaxin 1A, synaptosome-associated protein 25, and vesicle-associated membrane protein in the prefrontal and motor cortices between chronic alcoholics and control subjects. We found a region-specific elevation in synaptophysin I immunoreactivity in the prefrontal cortex of alcoholics, but detected no significant differences between the groups in the immunoreactivities of the other three proteins. Our findings are consistent with an effect of repeated ethanol exposure on modulators of synaptic strength but not on executors of glutamate release, and suggest a role for synaptophysin I in the enduring neuroplasticity in the prefrontal cortical glutamate circuitry that is associated with ethanol dependence.     

Prenatal stress‐related alterations in synaptic transmission and 5‐HT7 receptor‐mediated effects in the rat dorsal raphe nucleus are ameliorated by the 5‐HT7 receptor antagonist SB 269970

Early life adversity exerts a detrimental influence on developing brain neuronal networks and its consequences may include mental health disorders. In rats, prenatal stress may lead to anxiety and depressive‐like behavior in the offspring. Several lines of evidence implicated an involvement of prenatal stress in alterations of the brain serotonergic system functions, but the effects of prenatal stress on its core, the dorsal raphe nucleus (DRN), still remain incompletely understood. The present study was aimed at finding whether prenatal stress induces modifications in the glutamatergic and GABAergic inputs to DRN projection cells and whether it affects DRN 5‐HT₇ receptors, which modulate activity of these synapses. Prenatal stress resulted in an increase in basal frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and in a decrease in basal frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from putative projection neurons in DRN slices ex vivo. While there were no changes in the excitability of DRN projection neurons, the 5‐HT₇ receptor‐mediated reduction in the sEPSC frequency and rise in the sIPSC frequency, seen in control rats, were largely absent in slices obtained from prenatally stressed rats. Repeated administration of SB 269970, a 5‐HT₇ receptor antagonist, resulted in a reversal of prenatal stress‐induced alterations in 5‐HT₇ receptor‐mediated effects on the sEPSC/sIPSC frequency. Moreover, the treatment reversed prenatal stress‐induced alterations in basal excitatory transmission and partially reversed the effect of stress on basal inhibitory transmission in the DRN.     

Increased neuronal survival in the brainstem during liver injury: Role of γ‐aminobutyric acid and serotonin chitosan nanoparticles

γ‐Aminobutyric acid (GABA)‐ and serotonin (5‐HT)‐mediated cell signaling, neuronal survival enhancement, and reduced neuronal death in brainstem during liver injury followed by active liver regeneration have a critical role in maintaining routine bodily functions. In the present study, GABA_B and 5‐HT_2A receptor functional regulation, interrelated actions of neuronal survival factors, and expression of apoptotic factors in the brainstem during GABA and 5‐HT chitosan nanoparticles‐induced active liver regeneration in partially hepatectomized rats were evaluated. Partially hepatectomized rats were treated with the nanoparticles, and receptor assays and confocal microscopic studies of GABA_B and 5‐HT_2A receptors, gene expression studies of GABA_B and 5‐HT_2A receptors, nuclear factor‐κB (NF‐κB), tumor necrosis factor‐α (TNF‐α), Akt‐1, phospholipase C, Bax, and caspase‐8 were performed with the brainstems of experimental animals. A significant decrease in GABA_B and 5‐HT_2A receptor numbers and gene expressions denoted a homeostatic adjustment by the brain to trigger the sympathetic innervations during elevated DNA synthesis in the liver. The neuronal apoptosis resulting from the loss of liver function after partial hepatectomy was minimized by nanoparticle treatment in rats compared with rats with no treatment during regeneration. This was confirmed from the gene expression patterns of NF‐κB, TNF‐α, Akt‐1, phospholipase C, Bax, and caspase‐8. The present study revealed the potential of GABA and 5‐HT chitosan nanoparticles for increasing neuronal survival in the brainstem during liver injury following regeneration, which avoids many neuropsychiatric problems. © 2013 Wiley Periodicals, Inc.     

Modafinil enhances the increase of extracellular serotonin levels induced by the antidepressant drugs fluoxetine and imipramine: a dual probe microdialysis study in awake rat

In view of a postulated role of the vigilance-promoting drug modafinil in depression, the interaction of modafinil and two classical antidepressant drugs, fluoxetine and imipramine, were studied in 5-HT levels in the dorsal raphe-cortical system using dual-probe microdialysis. Fluoxetine (1-10 mg/kg) dose-dependently increased dorsal raphe-cortical 5-HT levels. Modafinil at a very low dose (3 mg/kg), by itself ineffective, enhanced the fluoxetine (5 mg/kg)-induced increases of 5-HT levels in both brain areas. A synergistic interaction was observed in the prefrontal cortex with fluoxetine (1 mg/kg) in terms of 5-HT release, but not in the dorsal raphe. Imipramine (1.3 mg/kg) increased 5-HT levels in the dorsal raphe, but not in the prefrontal cortex, while the higher doses (10.9-21.8 mg/kg) caused substantial increases in both brain areas. Modafinil (3 mg/kg), injected before imipramine (1.3 mg/kg), which by itself was ineffective on cortical 5-HT levels, increased cortical 5-HT levels. On other hand, modafinil failed to affect the high-dose imipramine (10.9 mg/kg)-induced increase of 5-HT levels in the prefrontal cortex and the imipramine (1.3; 10.9 mg/kg)-induced increase of 5-HT levels in the dorsal raphe nucleus. These results demonstrate that modafinil in low doses enhances the acute effects of fluoxetine and imipramine on 5-HT levels in the dorsal raphe nucleus (fluoxetine only) and especially in the prefrontal cortex of the awake rat. These findings suggest a therapeutic potential of low doses of modafinil in the treatment of depression when combined with low doses of classical antidepressants, especially by increasing 5-HT transmission in cortical regions.     

An immunocapture/scintillation proximity analysis of Gαq/11 activation by native serotonin (5‐HT)2A receptors in rat cortex: Blockade by clozapine and mirtazapine

Though transduction mechanisms recruited by heterologously expressed 5‐HT_2A receptors have been extensively studied, their interaction with specific subtypes of G‐protein remains to be directly evaluated in cerebral tissue. Herein, as shown by an immunocapture/scintillation proximity analysis, 5‐HT, the prototypical 5‐HT_2A agonist, DOI, and Ro60,0175 all enhanced [³⁵S]GTPγS binding to Gαq/11 in rat cortex with pEC₅₀ values of 6.22, 7.24 and 6.35, respectively. No activation of Go or Gs/olf was seen at equivalent concentrations of DOI. Stimulation of Gαq/11 by 5‐HT (30 μM) and DOI (30 μM) was abolished by the selective 5‐HT_2A vs. 5‐HT_2C/5‐HT_2B antagonists, ketanserin (pK_B values of 9.11 and 8.88, respectively) and MDL100,907 (9.82 and 9.68). By contrast, 5‐HT‐induced [³⁵S]GTPγS binding to Gαq/11 was only weakly inhibited by the preferential 5‐HT_2C receptor antagonists, RS102,221 (6.94) and SB242,084 (7.39), and the preferential 5‐HT_2B receptor antagonist, LY266,097 (6.66). The antipsychotic, clozapine, which had marked affinity for 5‐HT_2A receptors, blocked the recruitment of Gαq/11 by 5‐HT and DOI with pK_B values of 8.54 and 8.14, respectively. Its actions were mimicked by the “atypical” antidepressant and 5‐HT_2A receptor antagonist, mirtazapine, which likewise blocked 5‐HT and DOI‐induced Gαq/11 protein activation with pK_B values of 7.90 and 7.76, respectively. In conclusion, by use of an immunocapture/scintillation proximity strategy, this study shows that native 5‐HT_2A receptors in rat frontal cortex specifically recruit Gαq/11 and that this action is blocked by clozapine and mirtazapine. Quantification of 5‐HT_2A receptor‐mediated Gαq/11 activation in frontal cortex should prove instructive in characterizing the actions of diverse classes of psychotropic agent. Synapse 63:95–105, 2009. © 2008 Wiley‐Liss, Inc.     

Effect of different challenge doses after repeated citalopram treatment on extracellular serotonin level in the medial prefrontal cortex: in vivo microdialysis study

Aims: In order to elucidate the relevance between the delayed onset of clinical efficacy of selective serotonin re‐uptake inhibitors (SSRI) and extracellular 5‐HT levels in the medial prefrontal cortex, the present study compared the ability of low‐dose (3 mg/kg) and high‐dose (30 mg/kg) citalopram to increase extracellular 5‐HT levels in the medial prefrontal cortex following repeated citalopram treatment using in vivo microdialysis. Methods: An SSRI, citalopram, was given 10 mg/kg, s.c. twice daily for 6 days and once on the seventh day in rats. On the eighth day, rats received a single injection of citalopram (3 or 30 mg/kg s.c.), and extracellular 5‐HT levels were assessed in the medial prefrontal cortex of rats using in vivo brain microdialysis. Results: There was no significant difference in basal extracellular 5‐HT levels between the repeated citalopram group and the repeated saline group. The low‐challenge dose of citalopram (3 mg/kg) produced significantly greater increases (170–200% at each time point) in the repeated citalopram group than in the repeated saline group (150%). The high‐challenge dose of citalopram (30 mg/kg), however, increased extracellular 5‐HT levels by 200–250% of basal levels in the repeated citalopram group, which was similar to the increases in the repeated saline group. Conclusions: Repeated SSRI treatment enhances the effect of low‐dose SSRI on extracellular 5‐HT levels but not that of high‐dose SSRI.     

Effects of prenatal exposure to amphetamine in the medial prefrontal cortex of the rat

This study was designed to investigate the effects of prenatal exposure to amphetamine in the organization of the medial prefrontal cortex of the rat, by an evaluation of growth, morphometric and neurochemical parameters. Pregnant Wistar rats were given 10 mg/kg body weight/day of D-amphetamine sulfate, subcutaneously, from gestational days 8 to 22. Control groups of pregnant rats were injected with saline, pair-fed or non-manipulated; litters were culled to eight pups (four males and four females), weighed every other day until postnatal day 30 and every week until day 90. The Gompertz model was used to study body weight evolution and the estimated growth parameters were not significantly different in the experimental groups. At postnatal days 14 and 30, the volumes of the prefrontal cortex, the fraction of neuropile occupied by neurons and the number of neurons per unit surface area were determined. The number of neurons per unit volume of reference area was calculated using the stereological technique of the dissector. For neurochemical analysis, the medial prefrontal cortex was dissected to measure the concentration of dopamine, serotonin and their metabolites. The allometric relationship of forebrain/body growth pointed to a mechanism of sparing and compensatory growth in the amphetamine exposed group. The changes found in the number of neurons per unit volume at postnatal day 14 show a catch-up at postnatal day 30. A decrease in serotonin levels was found in the amphetamine group compared with the pair-fed control, which was reflected in the ratio of serotonin to its metabolite, 5-hydroxyindolacetic acid. These changes, whether permanent or transitory, raise the possibility that some of the effects of prenatal exposure to amphetamine may be due to modifications in the neurotransmitter levels of serotonin.     

Elevated levels of endocannabinoids and CB1 receptor-mediated G-protein signaling in the prefrontal cortex of alcoholic suicide victims.

BACKGROUND: Alcoholism is often comorbid with mood disorders and suicide. We recently reported an upregulation of CB(1) receptor-mediated signaling in the dorsolateral prefrontal cortex (DLPFC) of subjects with major depression who died by suicide. In the present study, we sought to determine whether the changes in depressed suicides would also be present in alcoholic suicides and whether the endocannabinoid (EC) system plays a role in suicide in alcoholism. METHODS: The density of CB(1) receptor and its mediated [(35)S]GTP gamma S signaling were measured in the DLPFC of alcoholic suicides (AS) (n = 11) and chronic alcoholics (CA) (n = 11). The levels of ECs were measured by a liquid chromatograph/mass spectrometry. RESULTS: The CB(1) receptor density was higher in AS compared with the CA group in the DLPFC. Western blot analysis confirmed a greater immunoreactivity of the CB(1) receptor in AS. The CB(1) receptor-mediated [(35)S]GTP gamma S binding indicated a greater signaling in AS. Higher levels of N-arachidonyl ethanolamide and 2-arachidonylglycerol were observed in the DLPFC of AS. CONCLUSIONS: The elevated levels of ECs, CB(1) receptors, and CB(1) receptor-mediated [(35)S]GTP gamma S binding strongly suggest a hyperactivity of endocannabinoidergic signaling in AS. EC system may be a novel therapeutic target for the treatment of suicidal behavior.     

Burst‐firing patterns in the prefrontal cortex underlying the neuronal mechanisms of depression probed by antidepressants

Major depressive disorder (MDD) is one of the leading causes of morbidity worldwide. Several antidepressants have been widely prescribed to treat patients with MDD. However, neuronal changes in brain function remain poorly understood. Based on the standard chronic mild stress (CMS) model of depression in mice, we investigated the neuronal mechanisms of the classic antidepressant, fluoxetine, and a new compound (termed YY‐23 in this study) derived from furostanol saponin. The results showed that both fluoxetine and YY‐23 normalized CMS‐induced depressive‐like behaviors. YY‐23 caused antidepressant‐like behaviors with a faster action than fluoxetine. In terms of in vivo neuronal activities, a CMS‐induced decrease in spontaneous firing in burst of medial prefrontal cortex pyramidal neurons rather than ventral tegmental area (VTA) was reversed by the chronic administration of fluoxetine and YY‐23. We also found that CMS‐induced deficits in the expression of prefrontal brain‐derived neurotrophic factor (BDNF) were also restored by chronically administering YY‐23 and fluoxetine. In addition, chronic administration of fluoxetine rather than YY‐23 resulted in an improvement of antidepressive‐like behavior and a change of burst firing of VTA in control‐housed animals, indicating that the pharmacological effects of YY‐23 were specific to CMS‐treated animals. Together, these data suggest that the burst‐firing patterns of pyramidal cells may be a neural biomarker of depressive‐like mice and antidepressant action. Furthermore, synaptic transmission and BDNF may contribute to the rapid antidepressant‐like effects on depression.     

Architectonic subdivision of the human orbital and medial prefrontal cortex

The structure of the human orbital and medial prefrontal cortex (OMPFC) was investigated using five histological and immunohistochemical stains and was correlated with a previous analysis in macaque monkeys [Carmichael and Price (1994) J. Comp. Neurol. 346:366-402]. A cortical area was recognized if it was distinct with at least two stains and was found in similar locations in different brains. All of the areas recognized in the macaque OMPFC have counterparts in humans. Areas 11, 13, and 14 were subdivided into areas 11m, 11l, 13a, 13b, 13m, 13l, 14r, and 14c. Within area 10, the region corresponding to area 10m in monkeys was divided into 10m and 10r, and area 10o (orbital) was renamed area 10p (polar). Areas 47/12r, 47/12m, 47/12l, and 47/12s occupy the lateral orbital cortex, corresponding to monkey areas 12r, 12m, 12l, and 12o. The agranular insula (areas Iam, Iapm, Iai, and Ial) extends onto the caudal orbital surface and into the horizontal ramus of the lateral sulcus. The growth of the frontal pole in humans has pushed area 25 and area 32pl, which corresponds to the prelimbic area 32 in Brodmann's monkey brain map, caudal and ventral to the genu of the corpus callosum. Anterior cingulate areas 24a and 24b also extend ventral to the genu of the corpus callosum. Area 32ac, corresponding to the dorsal anterior cingulate area 32 in Brodmann's human brain map, is anterior and dorsal to the genu. The parallel organization of the OMPFC in monkeys and humans allows experimental data from monkeys to be applied to studies of the human cortex.