Denervation supersensitivity to serotonin in rat forebrain: Single cell studies

To investigate the development of denervation supersensitivity to serotonin (5-hydroxytryptamine, 5-HT) in the amygdala (AMYG) and the ventral lateral geniculate nucleus (vLGN), single cell recordings, microiontophoretic, histochemical and biochemical techniques were used in the present study. 5-HT projections to the vLGN and the AMYG were destroyed by 5,7-dihydroxytryptamine (5,7-DHT, a relatively selective toxin for 5-HT neurons) injected directly into the lateral ventricle or the ascending 5-HT pathway in the ventromedial tegmentum area. Enhanced responsiveness of cells to the inhibitory effect of microiontophoretically applied 5-HT (ionto-5-HT) began to develop within 24 h and approached a maximum 7 days after 5,7-DHT pretreatment. In general, the time courses for the reduction in both the density of 5-HT fluorescent varicosities and synaptosomal 5-HT uptake activity paralleled the time course for the development of denervation supersensitivity to 5-HT. During the first 2 days after 5,7-DHT, the enhanced sensitivity was selective for 5-HT; responses to D-lysergic acid diethylamide (LSD), norepinephrine (NE) and gamma-aminobutyric acid (GABA) were unchanged. Seven or more days after 5,7-DHT there was a marked increase of the responsiveness of neurons in the vLGN and the AMYG to both 5-HT and LSD (a 5-HT agonist which is not a substrate for the high affinity 5-HT uptake system). At these later times, the responsiveness of cells in the AMYG to NE and to a lesser extent GABA was also increased. In contrast to the marked supersensitivity seen after 5,7-DHT induced denervation, chronic administration of parachlorophenylalanine, a 5-HT synthesis inhibitor, failed to induce 5-HT supersensitivity.     

Monoaminergic denervation of the rat hippocampus: Microiontophoretic studies on pre- and postsynaptic supersensitivity to norepinephrine and serotonin

The responsiveness of hippocampal CA₃ pyramidal neurons to microiontophoretic applications of serotonin (5-HT), norepinephrine (NE), γ-aminobutyric acid (GABA) and isoproterenol (ISO) was assessed in rats following 5,7-dihydroxy-tryptamine (5,7-DHT) and 6-hydroxydopamine (6-OHDA) pretreatments and bilateral locus coeruleus lesions. The intraventricular administration of 200 μg (free base) of 5,7-DHT and of 6-OHDA produced 89% and 93% decreases of 5-HT and NE respectively. None of these pretreatments modified the initial responsiveness to, or recovery from iontophoretic application of 5-HT. In 6-OHDA pretreated and locus-lesioned rats, the initial effectiveness of NE was not altered but its effect was markedly prolonged. However, there was no such prolongation of the effect of ISO which is not a substrate for the high affinity NE reuptake. The effect of GABA was not affected by these pretreatments. Acute pharmacological blockade of the NE reuptake with desipramine (5 mg/kg, i.p.) similarly induced a prolongation of the effect of iontophoretically applied NE, while fluoxetine (10 mg/kg, i.p.) a 5-HT reuptake blocker, failed to alter the recovery of pyramidal cells from iontophoretic application of 5-HT.

It is concluded that 5-HT denervation induces neither pre- nor postsynaptic types of supersensitivity in hippocampal pyramidal cells, contrasting with the previously shown supersensitivity of ventral lateral geniculate and amygdaloid neurons following 5-HT denervation. NE denervation fails to induce a postsynaptic type of supersensitivity but leads to a marked prolongation of the response to NE indicative of a presynaptic mechanism. These results underscore the necessity for regional studies of neurotransmitters and drug action.     

5-HT depletion with 5,7-DHT, PCA and PCPA in mice: differential effects on the sensitivity to 5-MeODMT, 8-OH-DPAT and 5-HTP as measured by two nociceptive tests

Depletion of 5-hydroxytryptamine (5-HT) in mice was produced by intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT, 80 micrograms) or by systemic injections of p-chloroamphetamine (PCA, 3 X 40 or 4 X 40 mg/kg), p-chlorophenylalanine (PCPA, 5 X 400 or 14 X 400 mg/kg) or combined PCA (3 X 40 mg/kg) + PCPA (11 X 400 mg/kg). Neither of the pretreatments altered nociception in the increasing temperature hot-plate test, whereas hyperalgesia was demonstrated in 5,7-DHT lesioned animals in the tail-flick test. 5,7-DHT-pretreatment enhanced the antinociceptive effect of the 5-HT agonists 5-methoxy-N,N-dimethyltryptamine (5-MeODMT), 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and 5-hydroxytryptophan (5-HTP). This effect was observed after 2, 5 and 8 days in the tail-flick test and after 5 and 8 days in the hot-plate test. However, pretreatment with PCPA or PCA failed to alter the antinociception elicited by the 5-HT agonists, although a tendency towards enhancement of antinociception was found after combined treatment with PCA and PCPA. It is suggested that the injection of 5,7-DHT induces denervation supersensitivity of post-synaptic 5-HT receptors. The lack of such supersensitivity after PCPA-pretreatment which induces similar 5-HT depletion to 5,7-DHT, may suggest that other factors than the absence of 5-HT may contribute to the development of denervation supersensitivity. Alternatively, the three 5-HT depleting agents may produce a qualitatively different reduction of 5-HT.     

Sexual behavior in male rats after intracerebral injection of 5,7-dihydroxytryptamine

Adult intact, or castrated testosterone propionate (TP, 150 μg/kg) treated male rats, were tested for masculine sexual behavior after having been injected with 5,7-dihydroxytryptamine (5,7-DHT, 4 μg/4 ml) intracerebrally either alone or in combination with systemic treatment with protriptyline, a noradrenaline (NA) re-uptake blocking agent. No changes were found in the sexual behavior of intact rats although the brain 5-HT levels were reduced to about one-third of their normal value. By contrast, there was a marked increase in the proportion of rats showing ejaculation patterns in the castrate + TP group after 5,7-DHT lesion than in the vehicle-injected group.Compared to the control group, the 5,7-DHT group showed a reduced uptake of [³H]5-HT and [³H]NA in the hypothalamus. Also the uptake of [³H]amines in the cerebral cortex was lowered although the difference did not attain statistical significance. A statistically significant relationship was found between the behavioral changes and the reduction of [³H]5-HT uptake in the hypothalamus while no such relationship was found between the NA uptake and the behavioral changes.Tistochemical analysis of the site of the 5,7-DHT injections showed that the unspecific damage (nerve cell loss, glial cell infiltration) involved a somewhat larger area in the 5,7-DHT group than in the controls. These unspecific lesions were, however, located outside the region of the large medial 5-HT bundle.The results support the hypothesis that 5-HT serves as a transmitter in the neural processes underlying masculine sexual behavior and, further, points to one component of the ascending 5-HT projections which innervates inter alia the hypothalamus as being of particular importance in this context.     

Serotonergic function of aging hippocampal CA3 pyramidal neurons: Electrophysiological assessment following administration of 5,7-dihydroxytryptamine in the fimbria-fornix and cingulum bundle

Serotonergic (5-hydroxytryptamine; 5-HT) neuro-transmission has been implicated in the regulation of cognitive function and this neurotransmitter system may underlie selective neuronal degeneration found in the aging hippocampus. Age-dependent changes in 5-HT function of hippocampal CA3 subfield pyramidal neurons were evaluated in female Fischer 344 rats (2 and 17 months) following denervation of the serotonergic afferents to the dorsal hippocampus using the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). Vehicle (ascorbic saline) or 5,7-DHT was administered bilaterally in the fimbria-fornix/cingulum bundle and dorsal pyramidal cell responses to microiontophoretic application of 5-HT, the 5-HT_1A agonist (±)-8-hydroxy-2-(di-N-propylamino) tetralin, the 5-HT_1A antagonist WAY 100,135 and N-methyl-D-aspartate were recorded at 3 weeks post-lesion. Independent of changes in sensitivity to the inhibitory effects of 5-HT with aging, the time to recovery of cell firing following application of 5-HT was significantly increased in the 18 month 5,7-DHT group compared to the 18 month Vehicle and 3 month 5,7-DHT groups (3.3- and 2.6-fold, respectively). These results demonstrate that serotonergic neurotransmission is altered with aging following a selective neurotoxic insult to the hippocampus. J. Neurosci. Res. 47:58–67, 1997. © 1997 Wiley-Liss, Inc.     

Compensatory responses in the aging hippocampal serotonergic system following neurodegenerative injury with 5,7-dihydroxytryptamine

This study utilized a multidisciplinary approach to examine injury-induced compensatory responses in the aging hippocampal serotonin transporter (5-HTT), a membrane protein implicated in a variety of neurodegenerative disorders. Age-dependent cellular, anatomical, and physiological changes of the 5-HTT were evaluated in female Fischer 344 rats (2 and 17 months) following denervation of the serotonergic afferents (fimbria-fornix and cingulum bundle) to the dorsal hippocampus using the neurotoxicant 5,7-dihydroxytryptamine (5,7-DHT). Seven days following 5,7-DHT administration, a uniform loss of the hippocampal 5-HTT immunoreactivity was observed in both age groups. However, at 21 days 5-HTT immunoreactivity in young 5,7-DHT-treated animals was similar to control levels, indicative of recovery, while older animals exposed to 5,7-DHT did not show recovery of hippocampal 5-HTT expression. 5-HTT binding site density, as determined by quantitative autoradiography ([3H]citalopram), supported the immunohistochemical results by demonstrating a recovery of 5-HTT binding sites in young, but not old animals, at 21 days following the lesion (P < 0.001). Furthermore, cellular electrophysiological function of hippocampal CA1 pyramidal neurons in 3- and 18-month-old F344 rats at 21 days following 5,7-DHT or vehicle treatment were assessed using in vivo microiontophoretic application of serotonin (5-HT). Independent of changes in sensitivity to the inhibitory effects of 5-HT application, the time to recovery of cell firing following application of 5-HT was significantly increased in the 18-month 5,7-DHT group compared to the 18-month vehicle and 3-month 5,7-DHT groups (60 and 59% increases, respectively; P < 0.05). Overall, these series of studies comprise a model which can be used to identify cellular events underlying both the formation of injury-induced compensatory processes in younger animals and the lack thereof with advancing age.     

Proposed animal model of attention deficit hyperactivity disorder

Dopamine (DA) neurons are implicated in the hyperlocomotion of neonatal 6-hydroxydopamine (6-OHDA)-lesioned rats, an animal model of attention deficit hyperactivity disorder (ADHD). Because serotonin (5-HT) neurons mediate some DA agonist effects, we investigated the possible role of 5-HT neurons on locomotor activity. Rats were treated at 3 days after birth with vehicle or 6-OHDA (134 μg ICV; desipramine pretreatment, 20 mg/kg IP, 1 h), and at 10 weeks with vehicle or 5,7-dihydroxytryptamine (5,7-DHT; 75 μg ICV; pretreatment with desipramine and pargyline, 75 mg/kg IP, 30 min), to destroy DA and/or 5-HT fibers. Intense spontaneous hyperlocomotor activity was produced in rats lesioned with both 6-OHDA and 5,7-DHT. Locomotor time in this group was 550 ± 17 s in a 600 s session, vs. 127 ± 13 s in the 6-OHDA group and <75 s in 5,7-DHT and intact control groups (p < 0.001). Oral activity dose-effect curves established that 5,7-DHT attenuated DA D₁ receptor supersensitivity and further sensitized 5-HT_2c receptors. Acute treatment with dextroamphetamine (0.25 mg/kg SC) reduced locomotor time in 6-OHDA+5,7-DHT-lesioned rats to 76 ± 37 s (p < 0.001). Striatal DA was reduced by 99% and 5-HT was reduced by 30% (vs. 6-OHDA group). Because combined 6-OHDA (to neonates) and 5,7-DHT (to adults) lesions produce intense hyperlocomotion that is attenuated by amphetamine, we propose this as a new animal model of ADHD. The findings suggest that hyperactivity in ADHD may be due to injury or impairment of both DA and 5-HT neurons.     

Modified coeruleo-cortical noradrenergic neurotransmission after serotonin depletion by PCPA: electrophysiological studies in the rat

To detect eventual modifications in the efficacy of the noradrenergic (NA) coeruleo-cortical system after serotonin (5-HT) depletion by parachlorophenylalanine (PCPA), three electrophysiological parameters were investigated in urethane-anesthetized rats which were treated for 2 days with daily injections of this inhibitor of 5-HT synthesis. 1) The spontaneous activity of locus coeruleus (LC) noradrenergic neurons showed a significant increase in PCPA-treated compared to control rats (4.3 vs. 2.6 Hz). 2) The sensitivity of NA autoreceptors was measured in the LC by the effect of intravenous administrations of clonidine or microiontophoretic applications of NA on spontaneous neuronal firing. In treated rats, clonidine and NA induced a lesser reduction of LC neuron firing than in the controls (27 vs. 75% decreases and 1,367 vs. 280 nC, respectively). 3) The responsiveness of cortical neurons to electrical stimulation of the LC was assessed by peristimulus time histograms in the dorsal fronto-parietal cortex. Following stimulation at 2 or 4 Hz, a majority of spontaneously firing cortical units was inhibited by electrical stimulation of the LC, but the percentage of such units was reduced and showed a decreased responsiveness after PCPA treatment. These findings suggest that following 5-HT depletion by PCPA, cortical NA neurotransmission is markedly reduced in its efficacy in spite of some increase in the spontaneous activity of coeruleo-cortical NA neurons.     

Hippocampal serotonin re-uptake and nocturnal locomotor activity after microinjections of 5,7-DHT in the fornix-fimbria

The role of the hippocampal 5-hydroxytryptamine (5-HT) terminals in the control of locomotor activity was investigated by lesioning 5-HT axons in the fimbria with 5,7-dihydroxytryptamine (5,7-DHT). Rats pretreated with desimipramine (10 mg/kg, i.p.) received microinjections of 5,7-DHT (0, 1, 3, 5 or 10 μg in 0.4 μl ascorbic Ringer's solution) into the fornix-fimbria. On the fourteenth to twenty-first nights after operation, nocturnal locomotor activity was measured in photocell cages. Twenty-eight to thirty days after operation degeneration of 5-HT terminals was assessed by measuring in vitro [³H]5-HT re-uptake in slices of dorsal hippocampus, ventral hippocampus and the septum.Groups injected with 5,7-DHT showed hyperactivity in the night period and increased decrements of activity between tests, both of which were related to the dose of neurotoxin. A reduction of [³H]5-HT re-uptake was found in dorsal hippocampus which was related to the dose of 5,7-DHT, but ventral hippocampal and septal [³H]5-HT re-uptake were not systematically reduced. For each rat, levels of dorsal and ventral hippocampal [³H]5-HT re-uptake were negatively correlated with the mean nocturnal activity from the 7 nights of testing. Levels of dorsal, but not ventral hippocampal [³H]5-HT re-uptake were negatively correlated with the mean nightly decrement of activity. No correlations were found between septal [³H]5-HT and these activity measures. These results, indicate that the increase in nocturnal locomotor activity caused by generalized depletion of 5-HT in the brain may be due to disruption of hippocampal 5-HT terminals supplied by the fornix-fimbria.     

Ascending afferent regulation of rat midbrain dopamine neurons

Standard, extracellular single-unit recording techniques were used to examine the electrophysiological and pharmacological responsiveness of midbrain dopamine (DA) neurons to selected, ascending afferent inputs. Sciatic nerve stimulation-induced inhibition of nigrostriatal DA (NSDA) neurons was blocked by both PCPA (5-HT synthesis inhibitor) and 5,7-DHT (5-HT neurotoxin), suggesting mediation by a serotonergic (5-HT) system. Direct stimulation of the dorsal raphe (which utilizes 5-HT as a neurotransmitter and inhibits slowly firing NSDA neurons) inhibited all mesoaccumbens DA (MADA) neurons tested. Paradoxically, DPAT, a 5-HT_1a agonist which inhibits 5-HT cell firing, enhanced sciatic nerve stimulation-induced inhibition of NSDA neurons. MADA neurons were not inhibited by sciatic nerve stimulation and, therefore, could not be tested in this paradigm. In contrast to the dorsal raphe, electrical stimulation of the pedunculopontine tegmental nucleus preferentially excited slowly firing NSDA and MADA neurons. Thus, both excitatory and inhibitory ascending afferents influence the activity of midbrain DA neurons, and intact 5-HT systems are necessary for sciatic nerve stimulation to alter DA cell activity. However, the role that 5-HT plays in mediating peripheral sensory input remains unclear.     

The functional significance of neonatal 5,7-dihydroxytryptamine lesions in the rat: response to selective 5-HT1A and 5-HT2,1C agonists

To study the involvement of serotonin (5-HT) receptor subtypes in behavioral supersensitivity following neonatal 5,7-dihydroxytryptamine (5,7-DHT) lesions, we measured acute behavioral responses to a single dose of selective 5-HT1A (8-OH-DPAT) or 5-HT2,1C (DOI) agonist compared to 5-hydroxytryptophan (5-HTP) in rats injected with 5,7-DHT intraperitoneally or intracisternally 14 weeks earlier. Only intraperitoneal 5,7-DHT injection resulted in brainstem 5-HT hyperinnervation, but cortical 5-HT depletions were also less. Effects of DOI, such as shaking behavior and forepaw myoclonus, were enhanced by 5,7-DHT lesions made intracisternally not intraperitoneally, whereas 8-OH-DPAT-evoked behaviors, such as forepaw myoclonus and head weaving, were enhanced more by the intraperitoneal route. The main consequence of intraperitoneal compared to intracisternal 5,7-DHT injection on supersensitivity to 5-HT agonists was increased presynaptic 5-HT1A responses and decreased 5-HT2,1C responses. In contrast, 5-HTP evoked more shaking behavior and less of the serotonin syndrome with the intraperitoneal compared to the intracisternal route of 5,7-DHT injection. Behavioral supersensitivity to 5-HTP, which was attributable to 5-HT1A, 5-HT2,1C, and possibly to other 5-HT receptors, was orders of magnitude greater than that elicited by direct receptor agonists and more clearly differentiated between rats with 5,7-DHT lesions and their controls, and between routes of 5,7-DHT injections, than responses to 5-HT agonists at the dose studied. 5,7-DHT induced dysregulation of 5-HT receptors, including both presynaptic and postsynaptic changes and altered interactions between receptor subtypes, better explains these data than postsynaptic changes alone.     

Serotonin neurons grafted to the adult rat hippocampus. II. 5-HT release as studied by intracerebral microdialysis

Extracellular levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were monitored by microdialysis in the hippocampal formation previously denervated of its serotonergic input by an intraventricular injection of 5,7-dihydroxytryptamine (5,7-DHT), and in 5,7-DHT denervated hippocampi reinnervated by grafted fetal rat serotonin neurons. Two weeks after 5,7-DHT lesion, baseline 5-HT release was reduced to levels below detection, and KCl- and p-chloro-amphetamine-evoked release was reduced by 90-95%. In the chronically denervated hippocampus (3 months after lesion), baseline 5-HT release had recovered to near-normal levels, but KCl- and p-chloroamphetamine-evoked release remained severely impaired. Addition of the 5-HT re-uptake blocker indalpine to the perfusion medium induced a 5-6-fold increase in serotonin overflow in the normal hippocampus, while the serotonin overflow in the 5,7-DHT denervated hippocampus remained unaffected. The intrahippocampal fetal raphe transplants restored 5-HT release to near-normal levels, not only under baseline conditions but also in the presence of re-uptake blockade. Both KCl- and p-chloroamphetamine-induced release had recovered in the grafted hippocampus and the responses were even greater than those seen in normal animals. In both normal and grafted hippocampus addition of the sodium channel blocker tetrodotoxin reduced 5-HT overflow to the level seen in the denervated hippocampus. The new hippocampal serotonin innervation, established by the grafts, was markedly denser than normal, and the tissue 5-HT and 5-HIAA levels were 3-4-fold higher than normal in the grafted hippocampi. The 5-HIAA level in the perfusate collected from the grafted hippocampi showed a similar increase above normal, whereas 5-HT release was maintained within the normal range, both under baseline conditions and in the presence of re-uptake blockade. The results indicate that the grafted serotonergic raphe neurons are spontaneously active at the synaptic level, despite their ectopic location. The ability of the grafted neurons to maintain 5-HT release within the normal range suggests that local regulatory mechanisms at the terminal level can compensate for abnormalities in the graft-derived innervation density.     

Intracisternal 5,7-dihydroxytryptamine lesions in neonatal and adult rats: comparison of response to 5-hydroxytryptophan

There have been few previous studies of the functional significance of 5,7-dihydroxytryptamine (5,7-DHT) lesions made in neonatal rats. To study the role of serotonin (5-HT) in recovery of function, rat pups and adult rats were injected intracisternally with 5,7-DHT or saline and challenged acutely with the 5-HT precursor 5-hydroxytryptophan (5-HTP) 4 weeks later as a test of behavioral supersensitivity. Compared to 5,7-DHT lesions in adults, neonatal lesions induced significantly greater 5-HT depletions in brainstem, but 5-HT depletions in other regions were not significantly different in the two groups. Rats with early 5,7-DHT lesions displayed supersensitive behavioral responses to 5-HTP, consisting of all the component myoclonic-serotonergic behaviors seen in rats with 5,7-DHT lesions made as adults. However, there was significantly less 5-HTP-evoked head weaving, truncal myoclonus and shaking behavior in rats treated with 5,7-DHT as neonates. Body weight was reduced both in rats with early and late 5,7-DHT lesions, but reduction persisted in rats with early lesions. These data indicate overall similarity with some differences between neurochemical and behavioral effects of early and late 5,7-DHT lesions made by the intracisternal route. They suggest that recovery mechanisms did not occur or failed to reverse the neurochemical or behavioral consequences of early 5,7-DHT lesions.     

Increased sensitivity to intracerebroventricular infusion of serotonin and deaminated indoles after lesioning rat with dihydroxytryptamine

Rats were prepared with a chronic intracerebroventricular cannula, and treated with intracisternal 5,7-dihydroxytryptamine (DHT) after i.p. desmethylimipramine or control vehicle. After recovery, they were tested behaviorally by direct observation and electronic monitoring of motor activity. Intraventricular infusion of a placebo or 5-hydroxyindoleacetic acid (5-HIAA) had little effect, but serotonin (5-HT) decreased, and norepinephrine increased locomotor activity in intact rats. Following pretreatment with 5,7-DHT, a small increase in locomotor activity was noted which was not altered by intracranial infusion of vehicle. In contrast, infusions of 5-HT produced a striking dose-dependent (ED₅₀ = 5 μg/min) pattern of hyperactivity, ‘myoclonic’ jerking movements, postural changes, and autonomic responses. Norepinephrine increased locomotor activity in the DHT-lesioned rats (but not significantly more than in controls), but failed to produce the myoclonic syndrome. The deaminated indoles, indoleacetaldehyde and 5-HIAA were more potent than 5-HT in producing the myoclonic response; tryptamine when infused at an equimolar dose had no effect. The putative serotonin antagonists, cyproheptadine and methiothepin (i.p.), were more effective in blocking responses to infused 5-HT than to equipotent doses of deaminated indoles. These behavioral responses may represent exaggerated excitatory effects mediated by serotonin in the brain stem and spinal cord, possibly modified by altered forebrain mechanisms. A neurophysiologic or neuropharmacologic role for deaminated indoles should be reconsidered as they may not merely be inactive metabolites.     

Brainstem serotonergic hyperinnervation modifies behavioral supersensitivity to 5-hydroxytryptophan in the rat

Rat pups were injected intracisternally (i.c.) or intraperitoneally (i.p.) with 5,7-dihydroxytryptamine (5,7-DHT) or saline and challenged 2 and 14 weeks later with the 5-HT precursor 5-hydroxytryptophan (5-HTP), which evokes behavioral supersensitivity in adult rats, 5,7-DHT induced transient postinjection convulsions in rats injected i.c. but not i.p. Rats with either type of 5,7-DHT lesions displayed supersensitive behavioral responses to 5-HTP. However, rats lesioned by i.p. injections exhibited significantly greater shaking behavior (+1445%) in response to 5-HTP than their i.c. counterparts, who instead showed more forepaw myoclonus (+250%) and head weaving (+270%), the core features of the 5-HT syndrome. Differences in 5-HT syndrome behaviors were already present 2 weeks after lesioning, whereas the difference in shaking behavior was not. After 14 weeks, 5-HT was selectively depleted (-43 to -92%) in hippocampus, spinal cord, and frontal cortex, and differences between i.c. and i.p. 5,7-DHT routes were insignificant except in frontal cortex. Brainstem 5-HT concentrations were significantly increased (+35%) after i.p. 5,7-DHT injections in contrast to reduction (-89%) after i.c. 5,7-DHT; 5-hydroxyindole acetic acid/5-hydroxytryptamine (5-HIAA/5-HT) ratios were decreased (-20%) with either route. These data suggest that brainstem 5-HT hyperinnervation following i.p. 5,7-DHT injection modifies the functional consequences of injury in abating the 5-HT syndrome, but does not result in complete recovery since shaking behavior is enhanced. Loss of presynaptically mediated autoregulation or receptor dysregulation may play a major role in behavioral supersensitivity induced by 5-HTP in rats with 5,7-DHT lesions. To the extent that the 5-HT syndrome is mediated by 5-HT1A receptors and shaking behavior by 5-HT2 sites, differential responses to injury of 5-HT1A and 5-HT2 receptors may contribute to these behavioral differences.     

Prefrontal cortex serotonin, stress, and morphine-induced nucleus accumbens dopamine

Uncontrollable, but not controllable, stress produces a persistent potentiation of morphine-induced nucleus accumbens dopamine (DA) efflux and morphine-induced medial prefrontal cortex serotonin (5-HT) efflux. Here we investigate medial prefrontal cortex 5-HT mediation of this potentiation. Male Sprague-Dawley rats received bilateral medial prefrontal cortex microinjections of the neurotoxin 5,7-dihydroxytriptamine (5,7-DHT, 8 microg/microl/side), which selectively depleted medial prefrontal cortex 5-HT, or vehicle (Sham), and cannula implantation in the nucleus accumbens shell. After 2 weeks, rats received either uncontrollable stress or no stress. Microdialysis and morphine (3 mg/kg) treatment were performed the following day. Morphine produced an enhanced increase in DA in the Stress-Sham group that was completely blocked by 5,7-DHT lesions, suggesting that 5-HT in the medial prefrontal cortex mediates this potentiation.     

Serotonin 2A receptor mRNA levels in the neonatal dopamine-depleted rat striatum remain upregulated following suppression of serotonin hyperinnervation.

Sixty days after bilateral dopamine (DA) depletion (>98%) with 6-hydroxydopamine (6-OHDA) in neonatal rats, serotonin (5-HT) content doubled and 5-HT(2A) receptor mRNA expression rose 54% within the rostral striatum. To determine if striatal 5-HT(2A) receptor mRNA upregulation is dependent on increased 5-HT levels following DA depletion, neonatal rats received dual injections of 6-OHDA and 5,7-dihydroxytryptamine (5,7-DHT) which suppressed 5-HT content by approximately 90%. In these 6-OHDA/5,7-DHT-treated rats, striatal 5-HT(2A) receptor mRNA expression was still elevated (87% above vehicle controls). Comparative analysis of 5-HT(2C) receptor mRNA expression yielded no significant changes in any experimental group. These results demonstrate that upregulated 5-HT(2A) receptor biosynthesis in the DA-depleted rat is not dependent on subsequent 5-HT hyperinnervation.     

Effects of intracisternal vs. intrahypothalamic 5,7-DHT on feeding elicited by hypothalamic infusion of NE.

A variety of evidence has led to suggestions that brain serotonin (5-HT) and norepinephrine (NE) interact within the medial hypothalamus to control food intake. To test the possibility that chronic decrements in 5-HT might enhance NE-induced feeding, adult male rats were prepared with permanently indwelling cannulae aimed at the paraventricular nucleus (PVN), then received either intracisternal (IC) or PVN injections of the 5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT) vs. its vehicle, 1% ascorbic acid. Over a 4-week period, IC-5,7-DHT rats showed no signs of enhanced daily feeding or drinking. However, in 40-min intake tests, feeding but not drinking was enhanced by injecting 20 nmol NE into the PVN commencing 2 weeks after neurotoxin treatment. Terminal monoamine assays confirmed that IC-5,7-DHT produced large (80-90%) depletions of brain regional 5-HT. A functional index of 5-HT terminal damage was also implied by the impaired short-term feeding responses IC-5,7-DHT rats showed to the systemic administration of the 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) when tested between 3 and 4 weeks after IC treatment. Over a comparable 4-week period, PVN-5,7-DHT rats also showed no tendencies to overeat or overdrink on a daily basis. However, in contrast to IC-5,7-DHT rats, they also showed no differences in their feeding or drinking responses to NE injections into the PVN. This was so despite reliable depletions of 5-HT in the hypothalamus (-28%) and hippocampus (-71%). These results support earlier work showing that neither widespread nor localized hypothalamic damage to brain 5-HT neurons produce chronic overeating. However, the data suggest that phasic enhancements of PVN NE activity may trigger enhanced feeding when there is widespread damage to brain 5-HT neurons, although the PVN does not appear to be the brain site mediating this effect.     

Loss of serotonin uptake sites and immunoreactivity in rat cortex after dexfenfluramine occur without parallel glial cell reactions

The frontal cortices of rats which received eitherd,l- ord-fenfluramine (DFEN) for 4 days were examined 18 h to 2 weeks following treatment for changes in synaptosomal uptake of serotonin (5HT), paroxetine binding, 5HT-immunoreactivity (5HT-IR), and both astrocytic (GFAP) and microglial markers. Additional rats received intracerebroventricular injections of the neurotoxin 5,7-dihydroxytryptamine (DHT). Consistent with previous reports,d,l- and DFEN produced dose-dependent losses of both 5HT uptake and paroxetine binding, and loss of 5HT-IR which coincided with an abnormal or ‘swollen’ appearance of immunoreactive axon processes. Recovery of these serotonergic indices was greatest following the lowest doses of DFEN, but was absent after 5,7-DHT treatment. No evidence for an increase in GFAP synthesis or microglial activation was observed in frontal cortices of rats treated with either DFEN or 5,7-DHT. We conclude that the presence of swollen 5HT-IR axons in the cortices of both the 5,7-DHT and DFEN groups is insufficient to trigger the glial responses often associated with neuronal degeneration. Thus, it remains to be determined if swollen 5HT-IR axons are a prelude to neurodegeneration, or whether they represent reversible changes in axonal immunochemistry associated with decreases in 5HT levels. The implications of the data for the clinical safety of DFEN are briefly discussed.     

Behavioural modification of bulbospinal serotonergic inhibition and morphine analgesia

Habituation to the stress of sham nociceptive testing enhances a rat's sensitivity to noxious thermal stimuli and reduces the antinociceptive effect of a subsequent acute dose of morphine. Since serotonin (5-hydroxytryptamine, 5-HT) mediates stress responses, experiments were designed to elucidate the role of 5-HT in these phenomena. Intrathecal methysergide or 5,7-dihydroxytryptamine (5,7-DHT) reduced baseline tail-flick latencies of novice rats to those of habituated animals. Morphine dose-response relationships were fitted to a 4 parameter sigmoidal function. Baseline latencies of novice animals were increased by 5-hydroxytryptophan (5-HTP) and reduced by parachlorophenylalanine (PCPA) in both reflex tests and in the hot-plate test, but latencies of habituated animals were unchanged by either treatment. In both reflex tests, the maximum effect due to morphine was increased by 5-HTP and reduced by PCPA in novice but not in habituated animals. We conclude that the serotonergic component of morphine's bulbospinal action represents the stress of the testing environment rather than an essential part of morphine's action.