Time course of expression and function of the serotonin transporter in the neonatal rat's primary somatosensory cortex

Immunocytochemical and autoradiographic techniques were employed to determine the time course of expression of the serotonin (5-HT) transporter (SERT) on thalamocortical afferents in the rat's primary somatosensory cortex (S-I), and to correlate this expression to the transient vibrissae-related patterning of 5-HT immunostaining previously described. In additional in vivo and in vitro experiments, 5-HT and 3 H-5-HT were applied directly to the cortices of untreated and 5,7-dihydroxytryptamine-treated (5,7-DHT) rats in order to determine the period during which SERT functions on thalamocortical axons to take up 5-HT. In postnatal rats, SERT immunohistochemistry revealed a somatotopic patterning in S-I that persisted until P-15, which is 6 days after the disappearance of the vibrissae-related 5-HT immunostaining. 3 H-citalopram autoradiography revealed a vibrissae-related pattern in layer IV of S-I until at least P-30. Following destruction of raphe-cortical afferents with 5,7-DHT on the day of birth, this binding pattern remained visible until at least P-25, indicating that SERT located on thalamocortical axons is responsible for the 3 H-citalopram patterning observed in S-I. Tissue from 5,7-DHT-treated rats that had 5-HT applied directly to their cortices revealed a normal vibrissae-related pattern of 5-HT immunostaining in S-I at P-7 and P-11 but only a faint pattern at P-13 and none at P-14. In addition, 3 H-5-HT injected directly into S-I labeled layer IV barrels at P-6 and P-12 but not at P-18. The results of these experiments demonstrate that SERT is expressed by thalamocortical afferents and remains functional long after the vibrissae-related 5-HT immunostaining in cortex disappears.     

Effects of alterations of the vibrissae-related organization of thalamocortical axons upon the organization and outgrowth of intracortical connections in the barrelfield of the rat

Previous studies have shown that intracortical projections in layer IV of the vibrissae representation of primary somatosensory cortex (S-I) are arrayed in a pattern complementary to that of thalamocortical axons (TCAs). Elevation of cortical serotonin (5-HT) in rats during the first postnatal week results in a transient disruption of the vibrissae-related pattern of TCAs and layer IV neurons in S-I. The present study examines the influence of elevated cortical 5-HT levels and the attendant loss of vibrissae-related TCA clusters on the organization of S-I intracortical connections. Cortical 5-HT was elevated in neonatal rats via chronic injections of clorgyline from birth until P-6. Animals were euthanized on P-6 or allowed to survive an additional 4 days without further clorgyline treatment. Distributions of TCAs and intracortical axons were assessed via application of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Di-I) and 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (Di-A) to the thalamic radiations and directly into the cortical barrelfield, respectively. Chronic administration of clorgyline resulted in a loss of the vibrissae-related organization of TCAs in layer IV of S-I. There was also a loss of the complementary pattern of intracortical projections in layer IV of this region. Discontinuation of clorgyline treatment resulted in a return of the vibrissae-related pattern of TCAs as well as the complementary pattern of intracortical projections. These results are consistent with the conclusion that the normal organization of intracortical projections in this region of S-I depends on the presence of the orderly array of TCAs.     

Effects of alterations of the vibrissae-related organization of thalamocortical axons upon the organization and outgrowth of intracortical connections in the barrelfield of the rat

Previous studies have shown that intracortical projections in layer IV of the vibrissae representation of primary somatosensory cortex (S-I) are arrayed in a pattern complementary to that of thalamocortical axons (TCAs). Elevation of cortical serotonin (5-HT) in rats during the first postnatal week results in a transient disruption of the vibrissae-related pattern of TCAs and layer IV neurons in S-I. The present study examines the influence of elevated cortical 5-HT levels and the attendant loss of vibrissae-related TCA clusters on the organization of S-I intracortical connections. Cortical 5-HT was elevated in neonatal rats via chronic injections of clorgyline from birth until P-6. Animals were euthanized on P-6 or allowed to survive an additional 4 days without further clorgyline treatment. Distributions of TCAs and intracortical axons were assessed via application of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Di-I) and 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (Di-A) to the thalamic radiations and directly into the cortical barrelfield, respectively. Chronic administration of clorgyline resulted in a loss of the vibrissae-related organization of TCAs in layer IV of S-I. There was also a loss of the complementary pattern of intracortical projections in layer IV of this region. Discontinuation of clorgyline treatment resulted in a return of the vibrissae-related pattern of TCAs as well as the complementary pattern of intracortical projections. These results are consistent with the conclusion that the normal organization of intracortical projections in this region of S-I depends on the presence of the orderly array of TCAs.     

Contributions of raphe-cortical and thalamocortical axons to the transient somatotopic pattern of serotonin immunoreactivity in rat cortex

Two experiments were carried out to evaluate the relative contributions of thalamocortical and raphe-cortical fibers to the transient somatotopically organized pattern of serotonin (5-HT) immunoreactivity that appears in the primary somatosensory cortex (SI) of rats during the first 2 weeks of life. In the first experiment, the specific 5-HT uptake inhibitors, fluoxetine and paroxetine, were administered systemically, animals were killed 3, 6, or 12 h later, and cortices evaluated for 5-HT immunoreactivity. Fluoxetine treatment had no appreciable effect on the density of 5-HT immunoreactivity in the cortex. Paroxetine treatment caused a reduction in 5-HT immunoreactivity which was maximal 6 h after administration. Examination of the cortices of these animals revealed a loss of very fine dust-like 5-HT immunoreactivity, but a vibrissae-related pattern remained visible in thicker fibers. In a second experiment, raphe-cortical fibers were destroyed by systemic administration of 5,7-dihydroxytryptamine on the day of birth. Six days after this manipulation, 5-HT was applied directly to the cortex in vivo and the animals were then killed and cortices processed to demonstrate 5-HT immunoreactivity. The cortices of these rats revealed a fine dust-like immunoreactivity organized in a somatotopic pattern, but only very few 5-HT-positive axons. The results of these experiments suggest that both raphe-cortical axons and thalamocortical fibers contribute to the patterned 5-HT immunoreactivity observed in SI of perinatal rats.     

Differential expression of acetylcholinesterase in the brainstem, ventrobasal thalamus and primary somatosensory cortex of perinatal rats, mice, and hamsters

Acetylcholinesterase (AChE) is transiently expressed by thalamocortical axons in the rat, and staining for this enzyme has been used extensively to study the development of thalamocortical projections. In the present study, patterns of AChE staining were compared in the trigeminal brainstem, thalami and primary somatosensory cortices of perinatal rats, mice, and hamsters. As previously reported, the ventral posteromedial nucleus (VPM) of rats showed dense AChE staining from P-0 at least through P-8. The ventral posterolateral nucleus (VPL) contained heavy AChE staining at least through P-60. In the cortex, there was also dense AChE staining which was organized somatotopically in patches similar to those observed with other methods such as cytochrome oxidase (CO) staining. However, by adulthood, AChE staining revealed a negative image of the CO staining pattern in lamina IV. In the mouse and hamster, there was dense AChE staining inVPL from P-0 through adulthood, but VPM was much less heavily stained for this enzyme. Moreover, the staining in VPL of mice was markedly reduced after transection of axons that travel to the thalamus in the medial lemniscus, suggesting that much of it was contained in these afferent fibers. In the cortices of both perinatal and adult mice and hamsters, AChE staining yielded a negative image of the somatotopically organized patches demonstrable with CO staining. This negative image was apparent by P-2 in the mouse and P-4 in the hamster. These results document a dramatic species difference with respect to the expression of AChE in the thalami and cortices of developing rodents. The differences between the patterns observed in rats vs mice and hamsters probably reflect the fact that cortical AChE in the latter species is not contained in thalamocortical afferents arising from either VPM or VPL.     

Is the retrograde axonal transport of 3H-5-HT a specific process of serotoninergic neurons?

The specificity of the retrograde axonal transport of 3H-serotonin (3H-5-HT) was radioautographically studied in the afferents to the olfactory bulb (O.B.). Injections of 3H-5-HT of different concentrations (10(-2), 10(-3), 10(-4) and 10(-5) M) were performed into the O.B. of catron pretreated rats. Following injection of 3H-5-HT (10(-2) M), a cytoplasmic perikaryal labeling was observed in the bulk of afferents to the O.B. (aminergic and non-aminergic neurons). When lower concentrations of 3H-5-HT (10(-5) M) were injected into the O.B., the retrograde labeling was only seen in the raphe dorsalis (RD) serotoninergic perikarya. The specificity of the uptake-retrograde transport of 3H-5-HT seems to depend on the selectivity of uptake by nerve terminals.     

De- and regeneration of the bulbospinal serotonin neurons in the rat following 5,6-or 5,7-Dihydroxytryptamine treatment

Summary In an attempt to determine the conditions which permit central 5-HT neurons to respond to a chemical injury of their axons by sprouting and regeneration, the pattern and time-course of recovery of 5-HT concentrations and regrowth of bulbospinal 5-HT axons were evaluated in rats subjected to intraventricular treatment with either 75 g 5,6- or 150 g 5,7-DHT. While 5,6-DHT treatment is followed by a significant recovery of 5-HT concentrations in the telodiencephalon, brainstem and upper part of the spinal cord within 3 months, there is no significant restoration of the severely depleted 5-HT levels in the telodiencephalon and spinal cord, and only limited recovery in 5-HT content of the brainstem preparation after 5,7-DHT.These differences conform to the observation of widespread and effective regrowth and regeneration of the bulbospinal 5-HT neurons in the 5,6-DHT treated lower brainstem and upper spinal cord but restricted and localized sprouting efforts in the 5,7-DHT treated lower medulla oblongata. This could be explained by a cell body near lesion of the non-terminal indoleamine axons by 5,7-DHT which results in a late retrograde, irreversible degeneration of most of the indoleamine pericarya from group B1 and many of group B3.It is concluded that the preservation of a critical length of the main axon and part of its collaterals is necessary for the neuron's survival, and that the individual pattern of the neuropil architecture of brain centres which are invaded by the axonal sprouts may significantly influence their growth characteristics and thus either favour or impede their chance to reestablish connections with their original effector. Aberrant, localized, intense sprouting of drug-damaged axons may in itself reflect the need of the neuron—deprived of most of its axonal tree—to reestablish its original total axonal length by multiple branching.Supported by grants from the Deutsche Forschungsgemeinschaft. The authors are indebted to Rolf Franck for his technical assistance.Supported by grants from the Swedish Medical Research Council (No. 04 X-3874 and 04 X-56).     

In vivo regulation of the serotonin-2 receptor in rat brain

Serotonin-2 (5-HT-2) receptors in brain were measured using [3H]ketanserin. We examined the effects of amitriptyline, an antidepressant drug, of electroconvulsive shock (ECS) and of drug-induced alterations in presynaptic 5-HT function on [3H]ketanserin binding to 5-HT-2 receptors in rat brain. The importance of intact 5-HT axons to the up-regulation of 5-HT-2 receptors by ECS was also investigated, and an attempt was made to relate the ECS-induced increase in this receptor to changes in 5-HT presynaptic mechanisms. Twelve days of ECS increased the number of 5-HT-2 receptors in frontal cortex. Neither the IC50 nor the Hill coefficient of 5-HT in competing for [3H]ketanserin binding sites was altered by ECS. Repeated injections of amitriptyline reduced the number of 5-HT-2 receptors in frontal cortex. Reserpine, administered daily for 12 days, caused a significant increase in 5-HT-2 receptors, but neither daily injections of p-chlorophenylalanine (PCPA) nor lesions of 5-HT axons with 5,7-dihydroxytryptamine (5,7-DHT) affected 5-HT-2 receptors. However, regulation of 5-HT-2 receptors by ECS was dependent on intact 5-HT axons since ECS could not increase the number of 5-HT-2 receptors in rats previously lesioned with 5,7-DHT. Repeated ECS, however, does not appear to affect either the high-affinity uptake of [3H]5-HT or [3H]imipramine binding, two presynaptic markers of 5-HT neuronal function. 5-HT-2 receptors appear to be under complex control. ECS or drug treatments such as reserpine or amitriptyline, which affect several monoamine neurotransmission systems including 5-HT, can alter 5-HT-2 receptors. While depleting 5-HT alone (5,7-DHT or PCPA) does not alter [3H]ketanserin binding to 5-HT-2 receptors, intact 5-HT axons are necessary for the adaptive up-regulation of the receptor following ECS.     

Serotonin (5-HT) transport in human platelets is modulated by Src-catalysed Tyr-phosphorylation of the plasma membrane transporter SERT.

BACKGROUND/AIM: platelets possess tightly regulated systems for serotonin (5-HT) transport. This study analysed whether the 5-HT transport mediated by the plasma-membrane transporter SERT is regulated by its Tyr-phosphorylation. METHODS: 5-HT transport was determined by filtration techniques, while immunoblotting procedures were adopted for detecting the Tyr-phosphorylation of SERT in human platelet fractions. RESULTS: 5-HT accumulation in platelets pre-treated with reserpine, which prevents the neurotransmitter transport into the dense granules, decreased upon cellular exposure to PP2 and SU6656, two structurally unrelated inhibitors of Src-kinases. By contrast, the protein Tyr-phosphatase inhibitor pervanadate increased the 5-HT accumulation. Anti-SERT immunostaining of the platelet fractions showed a major band displaying an apparent molecular mass of 50 kappaDa, indicating that, during the analytical procedure, SERT underwent proteolysis, which was counteracted by addition of 4 M urea in the cellular disrupting medium. The Tyr-phosphorylation degree of SERT immunoprecipitated from membrane extracts decreased by platelet treatment with SU6656 or PP2, and enhanced upon pervanadate treatment. The anti-SERT immunoprecipitates displayed anti-Src immunostaining and in vitro kinase activity towards a Src-specific peptide-substrate. Platelet treatment with PP2 or SU6656 also caused a decrease in the imipramine binding to platelets. It was concluded that the Src-mediated SERT Tyr-phosphorylation regulates the 5-HT transport by affecting the neurotransmitter binding sites.     

Serotonin (5-HT) transporter (SERT) function after graded destruction of serotonergic neurons

The degree of occupancy of the serotonin transporter (SERT) by selective serotonin reuptake inhibitors (SSRIs) appears to be critical in determining therapeutic response. To gain insight into the extent of occupancy required to alter serotonergic neurotransmission we used high-speed chronoamperometry to determine the extent of serotonergic destruction required to reduce the clearance of exogenously administered serotonin from extracellular fluid in the CA3 region of the hippocampus. Rats were pretreated with various doses of 5,7-dihydroxytryptamine to produce either a low, intermediate or high loss of SERTs. Clearance of 5-HT was reduced only in rats with > 90% loss of SERT. In these rats, there was also a trend for peak signal amplitudes to be greater. There was no significant difference in these parameters between the sham group and those with low or intermediate loss of SERTs. The SSRI, fluvoxamine, prolonged clearance of 5-HT in sham, low and intermediate groups, whereas there was no effect of fluvoxamine in those rats with > 90% loss of SERT. Functional loss of SERT activity occurs when destruction of serotonergic innervation is greater than 90% but serotonin clearance and efficacy of fluvoxamine is maintained with as few as one fifth of a full complement of SERTs.     

Ephrins regulate the formation of terminal axonal arbors during the development of thalamocortical projections

The development of connections between thalamic afferents and their cortical target cells occurs in a highly precise manner. Thalamic axons enter the cortex through deep cortical layers, then stop their growth in layer 4 and elaborate terminal arbors specifically within this layer. The mechanisms that underlie target layer recognition for thalamocortical projections are not known. We compared the growth pattern of thalamic explants cultured on membrane substrates purified from cortical layer 4, the main recipient layer for thalamic axons, and cortical layer 5, a non-target layer. Thalamic axons exhibited a reduced growth rate and an increased branching density on their appropriate target membranes compared with non-target substrate. When confronted with alternating stripes of both membrane substrates, thalamic axons grew preferentially on their target membrane stripes. Enzymatic treatment of cortical membranes revealed that growth, branching and guidance of thalamic axons are independently regulated by attractive and repulsive cues differentially expressed in distinct cortical layers. These results indicate that multiple membrane-associated molecules collectively contribute to the laminar targeting of thalamic afferents. Furthermore, we found that interfering with the function of Eph tyrosine kinase receptors and their ligands, ephrins, abolished the preferential branching of thalamic axons on their target membranes, and that recombinant ephrin-A5 ligand elicited a branch-promoting activity on thalamic axons. We conclude that interactions between Eph receptors and ephrins mediate branch formation of thalamic axons and thereby may play a role in the establishment of layer-specific thalamocortical connections.     

Vascular reactivity, 5-HT uptake, and blood pressure in the serotonin transporter knockout rat

The handling of serotonin [5-hydroxytryptamine (5-HT)] depends on the serotonin transporter (SERT). A SERT knockout (KO) rat is a useful model to test the hypothesis that SERT is the primary mechanism for arterial 5-HT uptake and to investigate the impact of SERT removal on blood pressure. Wild-type (WT) and KO rats were used to measure 5-HT content (plasma, raphe, aorta, carotid, and mesenteric artery), aortic isometric contraction, and blood pressure. HPLC supported the lack of circulating 5-HT in plasma (ng/ml plasma, WT, 310 +/- 96; and KO, 1.0 +/- 0.5; P < 0.05). Immunohistochemistry and Western blot analyses validated the presence of the SERT protein in the WT rats and a lesser expression in the KO rat. The aorta isolated from KO rats had a normal contraction to phenylephrine and norepinephrine and a normal relaxation to the endothelium-dependent agonist acetylcholine compared with the aorta from WT. In contrast, the potency of 5-HT was increased in the aorta from KO rats compared with WT rats [-log EC(50) (M); WT, 5.71 +/- 0.08; and KO, 6.7 +/- 0.18] and maximum contraction was reduced [%phenylephrine (10 muM) contraction, WT, 113 +/- 6%; and KO, 52 +/- 12%]. 5-HT uptake was reduced but not abolished in arteries of the KO compared with the WT rats. Diurnal mean arterial blood pressure, heart rate, and locomotor activity level of the KO rats were similar to the WT rats. These data suggest that there are other mechanisms of 5-HT uptake in the arteries of the rat and that although the absence of circulating 5-HT and/or SERT function sensitizes arteries to 5-HT, SERT dysfunction does not impair normal blood pressure.     

Serotonin availability in rat colon is reduced during a Western diet model of obesity

Constipation and slowed transit are associated with diet-induced obesity, although the mechanisms by which this occurs are unclear. Enterochromaffin (EC) cells within the intestinal epithelium respond to mechanical stimulation with the release of serotonin [5-hydroxytryptamine (5-HT)], which promotes transit. Thus our aim was to characterize 5-HT availability in the rat colon of a physiologically relevant model of diet-induced obesity. EC cell numbers were determined immunohistochemically in chow-fed (CF) and Western diet-fed (WD) rats, while electrochemical methods were used to measure mechanically evoked (peak) and steady-state (SS) 5-HT levels. Fluoxetine was used to block the 5-HT reuptake transporter (SERT), and the levels of mRNA for tryptophan hydroxylase 1 and SERT were determined by quantitative PCR, and SERT protein was determined by Western blot. In WD rats, there was a significant decrease in the total number of EC cells per crypt (0.86 ± 0.06 and 0.71 ± 0.05 in CF and WD, respectively), which was supported by a reduction in the levels of 5-HT in WD rats (2.9 ± 1.0 and 10.5 ± 2.6 μM at SS and peak, respectively) compared with CF rats (7.3 ± 0.4 and 18.4 ± 3.4 μM at SS and peak, respectively). SERT-dependent uptake of 5-HT was unchanged, which was supported by a lack of change in SERT protein levels. In WD rats, there was no change in tryptophan hydroxylase 1 mRNA but an increase in SERT mRNA. In conclusion, our data show that foods typical of a WD are associated with decreased 5-HT availability in rat colon. Decreased 5-HT availability is driven primarily by a reduction in the numbers and/or 5-HT content of EC cells, which are likely to be associated with decreased intestinal motility in vivo.     

Dissociation of the plasticity of 5-HT1A sites and 5-HT transporter sites

To study the early effects of neonatal 5,7-dihydroxytryptamine lesions on 5-hydroxytryptamine_1A (5-HT_1A) receptors, we measured regional [³H]8-OH-DPAT-labeled 5-HT_1A sites in binding assays and compared them to our previous studies of [³H]paroxetine-labeled 5-HT transporter sites during the first month in the same rats. While there were significant time- and dose-dependent effects of 5,7-DHT on 5-HT transporter sites, there were no significant changes in 5-HT_1A sites in cortex, hippocampus, diencephalon, brainstem, cerebellum, or spinal cord. 5,7-DHT lesions also did not alter the K_i of Gpp(NH)p at brainstem 5-HT_1A sites or the K_i of 5-HT in cortex or brainstem in the presence or absence of GTPS or Gpp(NH)p. There were significant regional differences between the density of 5-HT_1A sites and 5-HT transporter sites. The ontogeny of brainstem 5-HT_1A sites was a pattern of increases until three weeks postnatal, and 5,7-DHT lesions did not alter the ontogeny of 5-HT_1A sites. These data suggest differential plasticity of 5-HT_1A and 5-HT transporter binding sites during the first month after neonatal 5,7-DHT lesions.     

Development and plasticity of cortical areas and networks

The development of cortical layers, areas and networks is mediated by a combination of factors that are present in the cortex and are influenced by thalamic input. Electrical activity of thalamocortical afferents has a progressive role in shaping cortex. For early thalamic innervation and patterning, the presence of activity might be sufficient; for features that develop later, such as intracortical networks that mediate emergent responses of cortex, the spatiotemporal pattern of activity often has an instructive role. Experiments that route projections from the retina to the auditory pathway alter the pattern of activity in auditory thalamocortical afferents at a very early stage and reveal the progressive influence of activity on cortical development. Thus, cortical features such as layers and thalamocortical innervation are unaffected, whereas features that develop later, such as intracortical connections, are affected significantly. Surprisingly, the behavioural role of 'rewired' cortex is also influenced profoundly, indicating the importance of patterned activity for this key aspect of cortical function.     

Pioneers in the Ventral Telencephalon

OL-protocadherin is a member of the non-clustered-type protocadherin family. A recent study of ours showed that it is essential not only for growth of striatal axons but also for higher ordered neural circuit formation in the ventral telencephalon. The phenotype of OL-protocadherin-deficient mice is striking: several major neural pathways such as thalamocortical pathway, corticothalamic pathway, corticospinal pathway and strionigral pathway were misrouted and/or congested in the ventral telencephalon. Moreover, we detected abnormal patterning of putative guidance cues for thalamocortical axons such as the Nkx2.1+ cell domain and permissive "corridor" in the ventral telencephalon. Analyses of the expression pattern and phenotypes suggested that deficiency of striatal axons is the primary cause of these phenotypes. With these observations in mind, we proposed a novel hypothesis that proper growth of striatal axons is essential in patterning guidance cues and subsequent formation of neural circuits in the ventral telencephalon. This hypothesis will open a new possibility to reveal the unknown mechanism of neural circuit formation in the ventral telencephalon.     

Cerebral Markers of the Serotonergic System in Rat Models of Obesity and After Roux-en-Y Gastric Bypass

Food intake and body weight are regulated by a complex system of neural and hormonal signals, of which the anorexigenic neurotransmitter serotonin (5-hydroxytryptamine or 5-HT) is central. In this study, rat models of obesity and weight loss intervention were compared with regard to several 5-HT markers. Using receptor autoradiography, brain regional-densities of the serotonin transporter (SERT) and the 5-HT(2A) and 5-HT(4) receptors were measured in (i) selectively bred polygenic diet-induced obese (pgDIO) rats, (ii) outbred DIO rats, and (iii) Roux-en-Y gastric bypass (RYGB)-operated rats. pgDIO rats had higher 5-HT(4) and 5-HT(2A) receptor binding and lower SERT binding when compared to polygenic diet-resistant (pgDR) rats. The most pronounced difference between pgDIO and pgDR rats was observed in the nucleus accumbens shell (NAcS), a brain region regulating reward aspects of feeding. No differences were found in the 5-HT markers between DIO rats, chow-fed control rats, and DIO rats experiencing a weight loss. The 5-HT markers were also similar in RYGB and sham-operated rats except for a downregulation of 5-HT(2A) receptors in the NAcS. The higher receptor and lower SERT binding in pgDIO as compared to pgDR rats corresponds to what is reported in overweight humans and suggests that the dysfunctions of the 5-HT system associated with overeating or propensity to become overweight are polygenically determined. Our results support that the obesity-prone rat model has high translational value and suggests that susceptibility to develop obesity is associated with changed 5-HT tone in the brain that may also regulate hedonic aspects of feeding.     

Responsiveness of cortical neurons to serotonin after 5,7-DHT denervation or PCPA depletion

1. In adult rats under urethane anesthesia, a vast majority of spontaneously active neurons in the frontoparietal cortex undergo a prolonged depression of their firing rate upon microiontophoretic application of 5-HT. 2. In 5,7-DHT-deafferented cortex, this effect is of a longer duration (mean 14 min) than in controls (mean 5 min). Moreover, small ejection currents of 5-HT are sufficient to induce a prolonged depression of the firing rate. 3. In PCPA-pretreated rats, there are no changes in the responsiveness to 5-HT. 4. In control and PCPA-pretreated rats, blocking of the 5-HT reuptake with fluoxetine increases the duration of responses to 5-HT (mean 15 min), whereas small ejection currents remain without effect. 5. These data indicate that, in the cerebral cortex, denervation supersensitivity to 5-HT results primarily from the removal of 5-HT afferents, and not from depletion of their 5-HT content. The enhanced responsiveness to microiontophoresed 5-HT appears to be due to a suppression of reuptake mechanisms, mainly responsible for the prolongation of 5-HT effects, and to a modification of receptors on target cells, which accounts for their greater sensitivity to 5-HT.     

Serotonin fiber innervation of cerebellar cell suspensions intraparenchymally grafted to the cerebellum ofpcd mutant mice

One aspect of integration of implanted neurons into the neuronal circuitry of a defective host brain is the re-establishment of a host-to-graft afferent innervation. We addressed this issue by using the adult cerebellum of Purkinje cell degeneration (pcd) mutant mice, which lack virtually all Purkinje cells after postnatal day (P) 45. Purkinje cells constitute one of the cerebellar cell types being innervated by axons of raphé serotonin (5-HT) neurons. In normal mice, 5-HT-immunoreactive fibers are distributed to all cerebellar folia. Following Purkinje cell loss inpcd mice, cerebellar 5-HT-immunoreactive fibers persist. Cerebellar cell suspensions were prepared from embryonic day (E) 11–13 normal mouse embryos and were intraparenchymally grafted into the cerebellum ofpcd mutants either directly or after pre-treatment with 5, 7-dihydroxytryptamine (5,7-DHT) to selectively remove 5-HT cells of donor origin. The state of Purkinje cells and 5-HT axons was monitored in alternate sections by 28-kDa Ca²⁺-binding protein (CaBP) and 5-HT immunocytochemistry, respectively. Serotonin-immunoreactive axons were seen in the grafts from 5 to 32 days after transplantation. In some of the grafts which had not been pre-treated with 5,7-DHT, a small number of 5-HT-immunoreactive cell bodies was found, indicating that part of the 5-HT fiber innervation of the graft could actually derive from donor cells. On the other hand, in grafts pre-treated with 5,7-DHT, no 5-HT cell bodies were seen in the grafted cerebellum; 5-HT fibre innervation of the grafts occurred, but it appeared to be slightly less robust compared to situations of co-grafted 5-HT cell bodies. These findings suggest that host 5-HT fibers are able to provide afferent innervation to donor cerebellar tissue; the presence of co-grafted 5-HT cells may augment such an innervation.Special issue dedicated to Dr. Morris H. Aprison.