Hadassah Tamir

Serotonin 1A Receptors, Serotonin Transporter Binding and Serotonin Transporter mRNA Expression in the Brainstem of Depressed Suicide Victims

Suicide and depression are associated with reduced serotonergic neurotransmission. In suicides, there is a reduction in serotonin transporter (SERT) sites and an increase in postsynaptic 5-HT1A receptors in localized regions of the prefrontal cortex. In depression, there is a diffuse decrease in SERT binding throughout the dorsoventral extent of the prefrontal cortex. Serotonergic innervation of the prefrontal cortex arises predominantly from neurons in the brainstem dorsal raphe nucleus (DRN). We, therefore, examined postmortem SERT binding and mRNA expression, as well as 5-HT1A autoreceptor binding in the DRN of 10 matched pairs of controls and depressed suicide victims. The concentration of SERT sites, SERT mRNA, and 5-HT1A binding was not different between controls and suicides (p > .05). In the DRN of suicides, the volume of tissue defined by 5-HT1A binding was 40% smaller than controls. An index of the total number of 5-HT1A receptors (receptor binding × volume of receptor distribution) was 43.3% lower in the DRN of suicides, compared with controls. The suicide group had 54% fewer DRN neurons expressing SERT mRNA compared with controls. In the serotonin neurons that expressed the SERT gene, expression per neuron was greater in suicides. Less total 5-HT1A and SERT binding is consistent with results of in vivo studies in depression. Less feedback inhibition of serotonin DRN firing via 5-HT1A autoreceptors and enhancement of serotonin action due to less uptake of serotonin, is consistent with compensatory changes in response to hypofunction in depressed suicides.

Neuronal tryptophan hydroxylase mRNA expression in the human dorsal and median raphe nuclei: major depression and suicide.

Major depressive disorder (MDD) and suicide are associated with deficient serotonergic neurotransmission. Tryptophan hydroxylase (TPH) is the rate-limiting biosynthetic enzyme for serotonin. Previously, we reported elevated levels of TPH protein in the dorsal raphe nucleus (DRN) of depressed suicides and now examine expression of neuronal TPH2 mRNA in a cohort of matched controls and depressed suicides (n=11 pairs). DRN TPH2 mRNA was measured by densitometric analysis of autoradiograms from in situ hybridization histochemistry experiments. TPH2 mRNA is confirmed as the raphe-specific isoform of TPH in human brain, and is expressed in neurons throughout the anteroposterior extent of the DRN and median raphe nucleus (MRN). TPH2 mRNA expression correlates with TPH protein distribution in the DRN, and has a negative correlation with age. In drug-free suicides, TPH2 expression is 33% higher in the DRN and 17% higher in the MRN as compared to matched nonpsychiatric controls. Higher levels of TPH2 mRNA were found throughout the entire extent of the rostrocaudal axis of the DRN, and were not specific to any single subnucleus. Higher TPH2 mRNA expression may explain more TPH protein observed in depressed suicides and reflect a homeostatic response to deficient brain serotonergic transmission.

Attenuated 5‐HT1A receptor signaling in brains of suicide victims: involvement of adenylyl cyclase, phosphatidylinositol 3‐kinase, Akt and mitogen‐activated protein kinase

Positron emission tomography studies in major depression show reduced serotonin (5‐HT)1A receptor antagonist‐binding potentials in many brain regions including occipital cortex. The functional meaning of this observation in terms of signal transduction is unknown. We used postmortem brain samples from depressed suicide victims to examine the downstream effectors of 5‐HT1A receptor activation. The diagnosis was established by means of psychological autopsy using Diagnostic and Statistical Manual of Mental Disorders (DSM) III‐R criteria. Measurements of [35S]GTPγS binding to Gαi/o in the occipital cortex of suicide victims and matched controls revealed a blunted response in suicide subjects and a decrease in the coupling of 5‐HT1A receptor to adenylyl cyclase. No significant group differences were detected in the expression levels of Gαi/o, Gαq/11 or Gαs proteins, or in the activity of cAMP‐dependent protein kinase A. Studies of a parallel transduction pathway downstream from 5‐HT1A receptor activation demonstrated a decrease in the activity of phosphatidylinositol 3‐kinase and its downstream effector Akt, as well as an increase in PTEN (phosphatase and tensin homolog deleted on chromosome 10), the phosphatase that hydrolyzes phosphatidylinositol 3,4,5‐triphosphate. Finally, the activation of extracellular signal‐regulated kinases 1 and 2 was attenuated in suicide victims. These data suggest that the alterations in agonist‐stimulated 5‐HT1A receptor activation in depressed suicide victims are also manifest downstream from the associated G protein, affecting the activity of second messengers in two 5‐HT1A receptor transduction pathways that may have implications for cell survival.

Enteric Dopaminergic Neurons: Definition, Developmental Lineage, and Effects of Extrinsic Denervation

The existence of enteric dopaminergic neurons has been suspected; however, the innervation of the gut by sympathetic nerves, in which dopamine (DA) is the norepinephrine precursor, complicates analyses of enteric DA. We now report that transcripts encoding tyrosine hydroxylase (TH) and the DA transporter (DAT) are present in the murine bowel (small intestine > stomach or colon; proximal colon > distal colon). Because sympathetic neurons are extrinsic, transcripts encoding TH and DAT in the bowel are probably derived from intrinsic neurons. TH protein was demonstrated immunocytochemically in neuronal perikarya (submucosal >> myenteric plexus; small intestine > stomach or colon). TH, DA, and DAT immunoreactivities were coincident in subsets of neurons (submucosal > myenteric) in guinea pig and mouse intestines in situ and in cultured guinea pig enteric ganglia. Surgical ablation of sympathetic nerves by extrinsic denervation of loops of the bowel did not affect DAT immunoreactivity but actually increased numbers of TH-immunoreactive neurons, expression of mRNA encoding TH and DAT, and enteric DOPAC (the specific dopamine metabolite). The fetal gut contains transiently catecholaminergic (TC) cells. TC cells are the proliferating crest-derived precursors of mature neurons that are not catecholaminergic and, thus, disappear after embryonic day (E) 14 (mouse) or E15 (rat). TC cells appear early in ontogeny, and their development/survival is dependent on mash-1 gene expression. In contrast, the intrinsic TH-expressing neurons of the murine bowel appear late (perinatally) and are mash-1 independent. We conclude that the enteric nervous system contains intrinsic dopaminergic neurons that arise from a mash-1-independent lineage of noncatecholaminergic precursors.

Hippocampal Granule Neuron Number and Dentate Gyrus Volume in Antidepressant-Treated and Untreated Major Depression

Smaller hippocampal volume is reported in major depressive disorder (MDD). We hypothesize that it may be related to fewer granule neurons (GN) in the dentate gyrus (DG), a defect possibly reversible with antidepressants. We studied age-, sex-, and postmortem interval-matched groups: no major psychopathology (controls); unmedicated-MDD; and MDD treated with serotonin reuptake inhibitors (MDD*SSRI) or tricyclics (MDD*TCA). Frozen right hippocampi were fixed, sectioned (50 μm), immunostained with neuronal nuclear marker (NeuN), and counterstained with hematoxylin. GN and glial number, and DG and granule cell layer (GCL) volumes were stereologically estimated. Fewer GNs in the anterior DG were present in unmedicated-MDDs compared with controls (p=0.013). Younger age of MDD onset correlated with fewer GNs (p=0.021). Unmedicated-MDDs had fewer mid-DG GNs than MDD*SSRIs (p=0.028) and controls (p=0.032). Anterior GCL glial number did not differ between groups. Anterior/mid GCL volume was smaller in unmedicated-MDDs vs controls (p=0.008) and larger in MDD*SSRIs vs unmedicated-MDDs (p<0.001), MDD*TCAs (p<0.001), and controls (p<0.001). Anterior GCL volume and GN number (r=0.594, p=0.001), and mid DG volume and GN number (r=0.398, p=0.044) were correlated. Anterior DG capillary density correlated with GN number (p=0.027), and with GCL (p=0.024) and DG (r=0.400, p=0.047) volumes. Posterior DG volume and GN number did not differ between groups. Fewer GNs in unmedicated-MDD without fewer neuronal progenitor cells, as previously reported, suggests a cell maturation or survival defect, perhaps related to MDD duration. This may contribute to a smaller hippocampus and is potentially reversed by SSRIs. Postmortem studies are correlative and animal studies are needed to test implied causal relationships.

Mechanism of extracellular Ca2+ receptor-stimulated hormone release from sheep thyroid parafollicular cells

1 Expression of receptors to extracellular calcium enables parafollicular cells of the thyroid gland (PF cells) to release calcitonin (CT) and serotonin (5‐HT) in response to increased external Ca2+. Recently, a calcium‐sensing receptor (CaR), similar to the G protein‐coupled receptor for external Ca2+ cloned from parathyroid gland, was shown to be expressed in PF cells. Using a highly purified preparation of sheep PF cells, we have examined the electrical and biochemical processes coupling CaR activation to hormone release. 2 Whole‐cell recordings in the permeabilized‐patch configuration show that elevated extracellular Ca2+ concentration ([Ca2+]o) depolarizes these cells and induces oscillations in membrane potential. In voltage clamp, high [Ca2+]o activates a cation conductance that underlies the depolarization. This conductance is cation selective, with a reversal potential near −25 mV indicating poor ion selectivity. 3 The CaR expressed in these cells is activated by other multivalent cations with a rank order potency of Gd3+ > Ba2+ > Ca2+≫ Mg2+. The insensitivity of these cells to high external Mg2+ contrasts with the reported sensitivity of the cloned CaR from parathyroid. 4 Elevation of [Ca2+]o also stimulates increases in intracellular Ca2+ concentration ([Ca2+]o) and this effect is largely inhibited by the Ca2+ channel blocker nimodipine, indicating that L‐type voltage‐gated Ca2+ channels contribute to the response to elevated [Ca2+]o. 5 Elevated [Ca2+]o induces an inward current under conditions where the only permeant external cation is Ca2+, indicating that influx via the cation conductance is another source of the increases in [Ca2+]i. 6 Extracellular Ca2+ stimulates 5‐HT release with an EC50 of 1.5 mm. Nimodipine blocks 90% of the Ca2+ ‐induced 5‐HT release, while other inhibitors of voltage‐gated calcium channels had no effect. These data support an important role for L‐type Ca2+ channels in CaR‐induced hormone secretion. Although earlier studies indicate that high [Ca2+]o induces release of Ca2+ from intracellular stores, thapsigargin‐induced depletion of these stores did not affect secretion from these cells, indicating that Ca2+ influx is necessary and sufficient for the Ca2+ ‐induced 5‐HT secretion. 7 Inhibition of protein kinase C (PKC) using chelerythrine, staurosporine, or calphostin C inhibited Ca2+ ‐induced 5‐HT release by 50% while phorbol ester‐induced 5‐HT secretion was completely inhibited. Thus, PKC is an important component of the pathway linking CaR activation to hormone release. However, another as yet unknown second messenger also contributes to this pathway. 8 We tested the contribution of two different phospholipases to the CaR responses to determine the source of the PKC activator diacylglycerol (DAG). Selective inhibition of phosphatidylinositol‐specific phospholipase C (PI‐PLC) with U73122 had no effect on the response to elevated [Ca2+]o. However, pretreatment with D609, a selective inhibitor of phosphatidylcholine‐specific phospholipase C (PC‐PLC), inhibited Ca2+ ‐induced 5‐HT release to 50% of control indicating that phosphatidylcholine is a likely source of DAG in the response of PF cells to elevated [Ca2+]o.

Release of [3H]serotonin and its binding protein from enteric neurons

The release of [3H]5-HT and its binding protein, SBP, from the guinea pig enteric nervous system was analyzed. Release of both [3H]5-HT and [3H]NE from strips of longitudinal muscle with adherent myenteric plexus preloaded with the respective radioactive amine was evoked by high K+ and the ionophore X537A. However, Ca2+-dependence could not be shown for [3H]5-HT release by either agent or for [3H]NE release by X537A. However, Ca2+-dependence (as well as inhibition of release by high Mg2+ and tetrodotoxin) could be demonstrated for the release of radioactivity evoked by electrical field stimulation of everted segments of ileum preloaded by perfusion through the serosal lumen with [3H]5-HT. Light and electron microscopic radioautography revealed that the sources of released radioactivity were axons, especially axonal varicosities containing a mixture of small clear and large dense-cored vesicles. SPB, but not the cytosol marker protein, lactic dehydrogenase, was spontaneously released from the perfused everted ileum. A marked increase in SBP (but not LDH) release was provoked by electrical field stimulation at 10 Hz, and this increased release (but not the spontaneous release) was Ca2+-dependent. It is concluded that SBP and 5-HT are probably stored together, at least in part in vesicles, and that both can be released by exocytosis from depolarized axon terminals.

Stimulation-dependent regulation of the pH, volume and quantal size of bovine and rodent secretory vesicles

Trapping of weak bases was utilized to evaluate stimulus‐induced changes in the internal pH of the secretory vesicles of chromaffin cells and enteric neurons. The internal acidity of chromaffin vesicles was increased by the nicotinic agonist 1,1‐dimethyl‐4‐phenyl‐piperazinium iodide (DMPP; in vivo and in vitro) and by high K+ (in vitro); and in enteric nerve terminals by exposure to veratridine or a plasmalemmal [Ca2+]o receptor agonist (Gd3+). Stimulation‐induced acidification of chromaffin vesicles was [Ca2+]o‐dependent and blocked by agents that inhibit the vacuolar proton pump (vH+‐ATPase) or flux through Cl− channels. Stimulation also increased the average volume of chromaffin vesicles and the proportion that displayed a clear halo around their dense cores (called active vesicles). Stimulation‐induced increases in internal acidity and size were greatest in active vesicles. Stimulation of chromaffin cells in the presence of a plasma membrane marker revealed that membrane was internalized in endosomes but not in chromaffin vesicles. The stable expression of botulinum toxin E to prevent exocytosis did not affect the stimulation‐induced acidification of the secretory vesicles of mouse neuroblastoma Neuro2A cells. Stimulation‐induced acidification thus occurs independently of exocytosis. The quantal size of secreted catecholamines, measured by amperometry in cultured chromaffin cells, was found to be increased either by prior exposure to L‐DOPA or stimulation by high K+, and decreased by inhibition of vH+‐ATPase or flux through Cl− channels. These observations are consistent with the hypothesis that the content of releasable small molecules in secretory vesicles is increased when the driving force for their uptake is enhanced, either by increasing the transmembrane concentration or pH gradients.

Storage of serotonin and serotonin binding protein in synaptic vesicles.

We have used the newly introduced method of DeLorenzo & Freedman (1978) for isolating synaptic vesicles to determine if such vesicles contain both serotonin (5‐HT) and serotonin binding protein (SBP). Two fractions were obtained. A 55, 000 g fraction was morphologically heterogeneous and contained coated vesicles. A 135, 0000 vesicle (dia. 51.3 nm) fraction was homogeneous in ultra‐structure and contained no coated vesicles. The specific activity of SBP in this fraction was much higher than that in the supernatant. Unlike SBP, very little lactic dehydrogenase activity appeared in the 135, 000 g fraction. Qualitative and quantitative differences were observed between the polypeptide profiles of soluble proteins extracted from the vesicles and supernatant proteins on SDS gels. Therefore, entrapment of cytosol in the vesicles of the 135, 000 g fraction was minimal. The 5‐HT concentration of the 135, 000 g vesicles was 5.5 ng/mg protein and in the supernatant, 11.3 ng/mg protein. The ATP concentration in the 135, 000 g vesicle fraction was only 0.8 ng/mg Pr. Rabbit spinal cords were transected in order to determine if SBP is moved proximo‐distally in axons by rapid axonal transport as would be predicted for a constituent of synaptic vesicles. SBP accumulated above the cut at a rate consistent with fast transport (78 mm/day). SBP activity fell caudal to the point of transection and there was no evidence, such as an accumulation below the lesion, that might indicate retrograde transport of SBP. These experiments indicate that SBP is probably synthesized in the cell bodies of serotonergic neurons and some is rapidly transported down axons to be stored in terminals in vesicles.

Expression and developmental regulation of oxytocin (OT) and oxytocin receptors (OTR) in the enteric nervous system (ENS) and intestinal epithelium

Although oxytocin (OT) and oxytocin receptor (OTR) are known for roles in parturition and milk let‐down, they are not hypothalamus‐restricted. OT is important in nurturing and opposition to stress. Transcripts encoding OT and OTR have been reported in adult human gut, and OT affects intestinal motility. We tested the hypotheses that OT is endogenous to the enteric nervous system (ENS) and that OTR signaling may participate in enteric neurophysiology. Reverse transcriptase polymerase chain reaction confirmed OT and OTR transcripts in adult mouse and rat gut and in precursors of enteric neurons immunoselected from fetal rats. Enteric OT and OTR expression continued through adulthood but was developmentally regulated, peaking at postnatal day 7. Coincidence of the immunoreactivities of OTR and the neural marker Hu was 100% in the P3 and 71% in the adult myenteric plexus, when submucosal neurons were also OTR‐immunoreactive. Co‐localization with NeuN established that intrinsic primary afferent neurons are OTR‐expressing. Because OTR transcripts and protein were detected in the nodose ganglia, OT signaling might also affect extrinsic primary afferent neurons. Although OT immunoreactivity was found only in ∼1% of myenteric neurons, extensive OT‐immunoreactive varicosities surrounded many others. Villus enterocytes were OTR‐immunoreactive through postnatal day 17; however, by postnatal day 19, immunoreactivity waned to become restricted to crypts and concentrated at crypt‐villus junctions. Immunoelectron microscopy revealed plasmalemmal OTR at enterocyte adherens junctions. We suggest that OT and OTR signaling might be important in ENS development and function and might play roles in visceral sensory perception and neural modulation of epithelial biology. J. Comp. Neurol. 512:256–270, 2009.