Peter Kalén

Endogenous Release of Neuronal Serotonin and 5‐Hydroxyindoleacetic Acid in the Caudate‐Putamen of the Rat as Revealed by Intracerebral Dialysis Coupled to High‐Performance Liquid Chromatography with Fluorimetric Detection

Abstract: Extracellular levels of endogenous serotonin (5‐HT) and its major metabolite, 5‐hydroxyindoleacetic acid (5‐HIAA), were measured in the caudate‐putamen of anesthetized and awake rats using intracerebral microdialysis coupled to HPLC with fluorimetric detection. A dialysis probe (of the loop type) was perfused with Ringer solution at 2 μl/min, and samples collected every 30 or 60 min. Basal indole levels were followed for up to 4 days in both intact and 5,7‐dihydroxytryptamine (5,7‐DHT) lesioned animals. Immediately after the probe implantation, the striatal 5‐HT levels were about 10 times higher than the steady‐state levels that were reached after 7‐8 h of perfusion. The steady‐state baseline levels, which amounted to 22.5 fmol/30 min sampling time, remained stable for 4 days. In 5,7‐DHT‐denervated animals, the steady‐state levels of 5‐HT, measured during the second day after probe implantation, were below the limit of detection (<10 fmol/60 min). However, during the first 6h post‐implantation, the 5‐HT output was as high as in intact animals, which suggests that the high 5‐HT levels recovered in association with probe implantation were blood‐derived. As a consequence, all other experiments were started after a delay of at least 12 h after implantation of the dialysis probe. In awake, freely moving animals, the steady‐state 5‐HT levels were about 60% higher than in halothane‐anesthetized animals, whereas 5‐HIAA was unaffected by anesthesia. KCI (60 and 100 mM) added to the perfusion fluid produced a sharp increase in 5‐HT output that was eight‐fold at the 60 mM concentration and 21‐fold at the 100 mM concentration. In contrast, 5‐HIAA output dropped by 43 and 54%, respectively. In 5,7‐DHT‐lesioned animals, the KCl‐evoked (100 mM) release represented less than 5% of the peak values obtained for the intact striata. Omission of Ca2+ from the perfusion fluid resulted in a 70% reduction in baseline 5‐HT output, whereas the 5‐HIAA levels remained unchanged. High concentrations of tetrodotoxin (TTX) added to the perfusion medium (5‐50 μM) resulted in quite variable results. At a lower concentration (1 μM), however, TTX produceda 50% reduction in baseline 5‐HT release, whereas the 5‐HIAA output remained unchanged. The 5‐HT reuptake blocker, indalpine, increased the extracellular levels of 5‐HT sixfold when added to the perfusion medium (1 μM), and threefold when given intraperitoneally (5 mg/kg). By contrast, the 5‐HIAA level remained unaffected during indalpine infusion. Application of TTX (1 μM) under simultaneous 5‐HT uptake blockade induced a decrease in 5‐HT output by 62–71%. p‐Chloroamphetamine (2.5 mg/kg, i.p.) induced a 12‐fold increase in 5‐HT release and reduced the 5‐HIAA output by about 50%. The p‐chloroamphetamine‐induced increase in 5‐HT release was 10 times lower in the 5,7‐DHT‐denervated striatum. Pargyline (75 mg/kg, i.p.) increased the extracellular levels of 5‐HT 11‐fold within 6 h, and reduced the 5‐HIAA levels by 80%. The 5‐HT receptor agonist, 5‐methoxy‐N,N‐dimethyltryptamine (1 mg/kg, i.p.), produced an immediate reduction of about 50% in 5‐HT release and a small (11 %) decrease in 5‐HIAA output. It is concluded (1) that intracerebral microdialysis coupled to HPLC with fluorimetric detection provides a useful method for the study of extracellular 5‐HT and 5‐HIAA levels; (2) that steady‐state levels of 5‐HT and 5‐HIAA recovered in the dialysis perfusate are neuronally derived, but these steady‐state levels are reached only after a minimum of 7–8 h after probe implantation; (3) that changes in striatal extracellular levels of 5‐HT are closely related to changes in serotonergic synaptic activity; and (4) that extracellular levels of 5‐HIAA are a poor indicator of synaptic activity, and instead primarily reflect intraneuronal metabolism.

In vivo release of DOPA and dopamine from genetically engineered cells grafted to the denervated rat striatum

Fibroblastic 3T3 and endocrine RIN cells were genetically modified by infection with a recombinant retrovirus encoding the form I of human tyrosine hydroxylase (TH) and selection in tyrosine-free medium. These cells were grafted to rats unilaterally lesioned with 6-hydroxy-dopamine. Both cell types survived implantation into the striatum, expressed TH immunoreactivity, and as assessed by microdialysis 8-9 days after implantation, secreted high amounts of DOPA and/or dopamine into the surrounding host striatum. The modified 3T3 cells secreted large amounts of DOPA that was efficiently decarboxylated to dopamine by the host striatal tissue; the newly synthesized dopamine was stored only to a limited extent in the denervated striatum. The modified RIN cells synthesized dopamine that was stored intracellularly and released in a regulated fashion. The grafted DOPA-secreting cells produced 4-5 times higher extracellular dopamine levels than the dopamine-secreting cells, and they were more efficient in reducing apomorphine-induced rotation. No effect was observed with either cell type on amphetamine-induced turning behavior.

Regional differences in the regulation of dopamine and noradrenaline release in medial frontal cortex, nucleus accumbens and caudate-putamen: a microdialysis study in the rat

Dopamine (DA) and noradrenaline (NA) extracellular levels have been measured by microdialysis in the medial frontal cortex (MFC), nucleus accumbens (NAc) and caudate-putamen (CP) under baseline conditions in awake and halothane-anaesthetized rats, and after application of three types of stimuli which are likely to activate the brainstem catecholaminergic systems: mild stressors (handling and tail pinch), rewarded behavior (eating palatable food without prior food deprivation) and electrical stimulation of the lateral habenular nucleus. Changes were studied with and without uptake blockade (10 microM nomifensine in the perfusion fluid). The influence of calcium concentration (1.2 or 2.3 mM in the perfusion fluid) on DA and NA overflow was tested in some cases. Handling and tail pinch stimulated both DA and NA overflow in MFC, and enhanced NA overflow in NAc. By contrast, these mildly stressful stimuli had only marginal effects on DA overflow in NAc and no effects on either DA or NA overflow in CP. Eating behavior was accompanied by increased DA and NA overflow in MFC but had no effect in NAc. These regional differences were similar also when the manipulations were applied under uptake blockade, which indicates that the more pronounced changes seen in MFC did not simply reflect a more sparse innervation (i.e. lower density of uptake sites) in the MFC compared to the more densely innervated NAc and CP areas. Stimulation of the lateral habenula induced a 2-3-fold increase in NA overflow in both MFC, NAc and CP but had no consistent effect on DA overflow in any region. The effect on NA release was abolished by a transection of the ipsilateral fasciculus retroflexus (which carries the efferent output of the lateral habenula). The results show that the forebrain DA and NA projections to cortical and striatal targets are differentially regulated during ongoing behavior, that the mesocortical and mesostriatal DA systems respond quite differently to stressful and rewarding stimuli; and that the NA projection to MFC (like the dopaminergic one) is more responsive to stressful and rewarding stimuli than the ones innervating the striatum (NAc and CP). The results support the view that environmental stimuli evoking emotional arousal (whether aversive or non-aversive) are accompanied by increased DA and NA release above all in the MFC and only to a minor extent in limbic and striatal areas.

Hippocampal Noradrenaline and Serotonin Release over 24 Hours as Measured by the Dialysis Technique in Freely Moving Rats: Correlation to Behavioural Activity State, Effect of Handling and Tail‐Pinch

Hippocampal extracellular levels of noradrenaline (NA), 5‐hydroxytryptamine (5‐HT), and 5‐hydroxyindole‐acetic acid (5‐HIAA) were monitored with the microdialysis technique in freely moving rats. In one experiment 30 min samples were collected during 24 h of continuous perfusion, and the monoamine output was compared to the behavioural activity state, as arbitrarily classified in three categories: sleep/rest, drowsiness and full alertness associated with complex behaviours. In the individual animal the hippocampal NA and 5‐HT output showed pronounced fluctuations during the 24 h period, but the 30 min sampling times did not allow for a clear‐cut correlation to behavioural activity state. However, the mean NA and 5‐HT output for all animals during the dark period of the day was 43 and 38% higher, respectively, than during the light period, and the average NA and 5‐HT levels in samples collected during periods of high behavioural activity was 34 and 45% higher, respectively, than during periods of rest or sleep. In contrast, there were no detectable changes in extracellular 5‐HIAA. The selective serotonin uptake blocker indalpine, added to the perfusion fluid at 1 μM, increased the extracellular 5‐HT levels 6‐fold, with a similar correlation to behavioural activity state as without indalpine.

Acetylcholine release in the rat hippocampus as studied by microdialysis is dependent on axonal impulse flow and increases during behavioural activation

Changes in extracellular levels of acetylcholine and choline in the hippocampal formation were measured using intracerebral microdialysis coupled to high performance liquid chromatography with post-column enzyme reaction and electrochemical detection. Various pharmacological and physiological manipulations were applied to awake unrestrained normal rats and rats subjected to a cholinergic denervation of the hippocampus by a complete fimbria-fornix lesion (1-2 weeks previously). Low baseline levels of acetylcholine (about 0.3 pmol/15 min sample) could be detected in the absence of acetylcholinesterase inhibition in all animals. However, in order to obtain stable and more readily detectable levels, the acetylcholinesterase inhibitor neostigmine was added to the perfusion medium at a concentration of 5 or 10 microM and was used during all subsequent manipulations. Addition of neostigmine increased acetylcholine levels approximately 10-fold (to 3.7 pmol 15 min) in the normal rats, which was about 4-fold higher than the levels recovered from the denervated hippocampi. Depolarization by adding KCl (100 mM) to the perfusion fluid produced a 3-fold increase in the extracellular acetylcholine levels, and the muscarinic antagonist atropine (3 microM) resulted in a 4-fold increase in the normal rats, whereas these drugs induced only small responses in the denervated rats. Neuronal impulse blockade by tetrodotoxin (1 microM) resulted, in normal rats, in a 70% reduction in extracellular acetylcholine levels. Sensory stimulation by handling increased acetylcholine levels by 94% in the normal rats, whereas this response was almost totally abolished in the denervated hippocampi. Behavioural activation by electrical stimulation of the lateral habenula resulted in a 4-fold increase in acetylcholine release in normal animals, and this response was totally blocked by a transection of the lateral habenular efferents running in the fasciculus retroflexus. The levels obtained by lateral habenula stimulation were reduced by about 95% in the rats with fimbria-fornix lesions. Following an acute knife transection of the fimbria-fornix performed during ongoing dialysis, acetylcholine levels dropped instantaneously by 70%, indicating that the extracellular acetylcholine levels in the hippocampus are maintained by a tonic impulse flow in the septohippocampal pathway. The extracellular levels of choline were reduced by about 30% after the addition of neostigmine in the normal rats, and increased by about 50% in both normal and denervated rats after addition of KCl to the perfusion fluid. No changes could be detected after atropine, handling, lateral habenula stimulation, or acute fimbria-fornix or fasciculus retroflexus transection.(ABSTRACT TRUNCATED AT 400 WORDS)

Possible excitatory amino acid afferents to nucleus raphe dorsalis of the rat investigated with retrograde wheat germ agglutinin and d-[3H]aspartate tracing

Evidence for excitatory amino acid afferents to nucleus raphe dorsalis (NRD) has been found with retrograde tracing techniques. For neuroanatomical definition of afferent sources to NRD, rats received stereotaxic injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) or implantations of crystal WGA-HRP in glass micropipettes. Retrogradely transported WGA-HRP was visualized with the tetramethyl-benzidine method, and afferents to NRD were identified from 20 different brain regions. Large numbers of labeled cells appeared in the lateral hypothalamus, lateral habenular nucleus, ventral tegmental area, periaqueductal gray, parabrachial nuclei and nucleus raphe magnus. Important inputs were also noted from dorsomedial hypothalamus and the area surrounding the perihypoglossal nucleus. Smaller numbers of WGA-HRP labeled cells appeared in bed nucleus of stria terminalis, diagonal band of Broca, cuneiform nucleus, superior vestibular nucleus, pontine periventricular gray, and some hypothalamic and reticular areas. Another group of rats received microinjections of D-[3H]aspartate (D[3H]Asp) and autoradiography consistently revealed retrograde labeling of cell bodies in 4 of the regions indicated by the WGA-HRP experiments as afferents to NRD. The most prominent aggregation of D-[3H]Asp-labeled cells was found in the lateral habenular nucleus, indicating that this input operates with an excitatory amino acid as transmitter. Significant numbers of D-[3H]Asp-labeled cells were also found in substantia nigra, periaqueductal and pontine periventricular gray. After large D-[3H]Asp injections involving NRD as well as surrounding areas, labeled cells were observed in several additional areas. Some of these areas were considered as afferents to surrounding periaqueductal gray or dorsal tegmental nuclei, while others may represent NRD afferents with relatively lower affinity for D-[3H]Asp. Several afferents to NRD failed to label with D-[3H]Asp, including diagonal band of Broca, hypothalamic areas, ventral tegmental area, parabrachial nuclei, locus coeruleus and reticular areas.

Afferents to the median raphe nucleus of the rat: Retrograde cholera toxin and wheat germ conjugated horseradish peroxidase tracing, and selectived-[3H]aspartate labelling of possible excitatory amino acid inputs

Afferents to the median-paramedian raphe nuclear complex, which contains the B8 serotonergic cell group, were investigated in the rat with neuroanatomical and transmitter-selective retrograde labelling techniques. Injection of sensitive retrograde tracers, cholera toxin genoid or wheat germ agglutinin conjugated horseradish peroxidase into the median raphe resulted in labelling of neurons in a large number of brain regions. Projections from 26 of these regions are supported by available orthograde tracing data; the cingulate cortex, bed nucleus of stria terminalis, medial septum and diagonal band of Broca, ventral pallidum, medial and lateral preoptic areas, lateral hypothalamus, dorsomedial nucleus of hypothalamus, lateral habenula, interpeduncular nucleus, substantia nigra, central (periaqueductal) gray, and laterodorsal tegmental nucleus seem to represent major sources of afferents to the median-paramedian raphe complex. Retrogradely labelled cells were also observed in a number of regions for which anterograde tracing data are not available, including the perifornical hypothalamic nucleus, ventral premammillary nucleus, supramammillary and submammillothalamic nuclei and the B9 area. Possible excitatory amino acid afferents were identified with retrograde D-[3H]aspartate labelling. Microinjection of D-[3H]aspartate at a low concentration, 10(-4) M in 50 nl, resulted in retrograde labelling of a limited number of median raphe afferents. The most prominent labelling was observed in the lateral habenula and the interpeduncular nucleus, but retrogradely labelled cells were also noted in the medial and lateral preoptic areas, lateral and dorsal hypothalamus, ventral tegmental area, laterodorsal tegmental nucleus, medial parabrachial nucleus, and the pontine tegmentum. After injections of 10(-3) M D-[3H]aspartate selective labelling also appeared in more distant afferent regions, including cells in cingulate cortex, and in some regions located at shorter distances, such as the supramammillary nucleus. Injections of D-[3H]aspartate at high concentration, 10(-2) M, resulted in the appearance of weakly to moderately labelled cells in most afferent areas which were devoid of labelled cells after injections of lower concentrations, suggesting that this labelling may be non-specific. It was concluded that the median-paramedian raphe receives afferents from a large number of forebrain and hypothalamic regions, while relatively few brain stem regions project to this nuclear complex. The selectivity of retrograde labelling with D-[3H]aspartate was found to be concentration dependent, and it is suggested that the connections showing high affinity for D-[3H]aspartate may use excitatory amino acids as transmitters. Excitatory amino acid inputs from lateral habenula and interpeduncular nucleus may play predominant roles in the control of ascending serotonergic and non-serotonergic projections originating in the median and paramedian raphe nuclei.

Dopaminergic transplants normalize amphetamine- and apomorphine-induced Fos expression in the 6-hydroxydopamine-lesioned striatum

Dopamine receptor-mediated Fos protein expression in the striatum has been used to monitor dopamine receptor activation at the cellular level after dopaminergic denervation and reinnervation by fetal nigral transplants. The pattern of striatal Fos expression after systemic administration of either the dopamine receptor agonist, apomorphine, or the dopamine-releasing agent, amphetamine, was studied in rats which had received cell suspension grafts of fetal ventral mesencephalic neurons into the striatum after a complete 6-hydroxydopamine lesion of mesostriatal dopaminergic projection. Grafted animals, and normal and lesioned controls were killed 2 h after administration of either D-amphetamine (5 mg/kg, i.p.) or apomorphine (0.25 mg/kg, s.c.). Fos protein was detected immunohistochemically, and the density of Fos-immunoreactive cell nuclei was measured in 12 selected areas of caudate-putamen, nucleus accumbens and globus pallidus by computerized image analysis. Consistent with previous studies, amphetamine induced high Fos expression in the medial and dorsal parts of the intact caudate-putamen and significantly lower expression in the denervated caudate-putamen. A significant difference between lesioned and intact striata was present also in globus pallidus, but not in nucleus accumbens. In grafted rats, amphetamine-induced Fos activation was restored to normal or supranormal levels in the anterior and central caudate-putamen (i.e. close to the graft deposits), whereas in the tail of caudate-putamen Fos expression was significantly lower than normal. The side-to-side difference in globus pallidus seen in lesioned rats was no longer present in the grafted animals. Apomorphine led to high Fos activation throughout the dopamine-depleted caudate-putamen, whereas only very few immunopositive cells were observed in the intact caudate-putamen. Also in globus pallidus and nucleus accumbens, a significantly higher number of Fos-immunoreactive cells was detected on the denervated side. In the grafted rats, apomorphine-induced Fos activation was similar to normal in all striatal areas sampled, as well as in the globus pallidus. The graft-induced effect extended over a considerably larger area than that covered by the graft-derived tyrosine hydroxylase-immunoreactive innervation. These findings indicate that fetal ventral mesencephalic transplants normalize dopamine receptor-mediated function in the 6-hydroxydopamine-lesioned caudate-putamen and nucleus accumbens, as well as in a primary target of the striatal output neurons, the globus pallidus. The results support the idea that dopamine released from the grafted neurons, both under baseline conditions and after amphetamine administration, exerts functional effects over a larger volume of the striatum than that reached by the graft-derived fibers.

Autoradiographic and electrophysiological evidence for excitatory amino acid transmission in the periaqueductal gray projection to nucleus raphe magnus in the rat

Selective retrograde labelling was used as an autoradiographic method to identify possible excitatory amino acid afferents to nucleus raphe magnus (NRM). Injections of 25-50 nl 10(-2) or 10(-3) M D-[3H]aspartate into the NRM resulted in prominent labelling of cells in ventrolateral mesencephalic periaqueductal gray (PAG). Electrophysiologically, stimulation in ventrolateral PAG excited cells in NRM with a latency of 2-12 ms. With many cells, microelectrophoretic application of the excitatory amino acid antagonists, kynurenate and gamma-D-glutamyl-glycine, resulted in a reversible reduction of the PAG-evoked response. Selective antagonists of N-methyl-D-aspartate (NMDA) were less effective. It is suggested that neurones in the ventrolateral PAG projecting to NRM utilize an excitatory amino acid or structurally related compound as a transmitter, and that this transmitter acts on receptors of the non-NMDA type.

Characterization of GABA release from intrastriatal striatal transplants: dependence on host-derived afferents.

Extracellular levels of GABA, derived from cell suspension transplants of embryonic day 14-15 rat striatal primordia implanted into the previously excitotoxically lesioned striatum, were measured using intracerebral microdialysis in halothane-anaesthetized rats. GABA overflow was monitored using loop type dialysis probes implanted into grafted, age-matched ibotenic acid-lesioned and intact striata, under baseline conditions and after different pharmacological manipulations. Basal and evoked GABA release, which was reduced by 58 and 96%, respectively, in the excitotoxin-lesioned striatum, was restored by the striatal grafts to levels close to or above those observed in normal striata. The graft-derived release of GABA was most likely of neuronal origin, since the K(+)-evoked (100 mM) GABA overflow was reduced by almost 80% when Ca++ was replaced by 20 mM Mg++ in the perfusion medium, and blockade of GABA uptake by nipecotic acid (0.5 mM), induced a greater than six-fold increase in GABA overflow. However, perfusion of the graft with 1 microM tetrodotoxin in combination with K+ (100 mM) resulted in little if any reduction in the K(+)-evoked overflow. Histological analysis demonstrated a dense tyrosine hydroxylase-positive fibre network in the grafts, which was removed after a 6-hydroxydopamine lesion of the ipsilateral nigrostriatal pathway. The dopamine denervating lesion resulted in an increased K(+)-evoked GABA overflow both in the intact (+76%) and the grafted striata (+181%), suggesting that the tonic dopaminergic inhibitory control of GABA release, seen in the intact striatum, is also present in the grafted striata. The glutamate analogue, kainic acid (1 mM added to the perfusion fluid), evoked a 60-74% increase in GABA overflow both in intact striata (with or without dopaminergic denervation) and in the striatal grafts. This effect seemed to be dependent on an intact corticostriatal projection, since knife-cut transections of the frontal cortex at the level of the forceps minor, abolished the response in both the intact and grafted striata. These results demonstrate that grafts of fetal striatal tissue implanted into the excitotoxically lesioned striatum restore striatal GABA overflow in a neuron-dependent manner, close to or above that seen in the normal intact striatum. Furthermore, the graft-derived GABA release appears to be under normal regulatory control from the host dopaminergic and glutamatergic systems. Since the GABAergic striatal output system is critical for the expression of striatum-related behaviours, it is proposed that the graft-induced behavioural recovery in the striatal lesion model, at least in part, may depend on the restoration of striatal GABAergic neurotransmission.