Cerebrospinal fluid γδ T cell frequency is age-related: a case-control study of 435 children with inflammatory and non-inflammatory neurological disorders.

Studies of cerebrospinal fluid (CSF) γδ T cells in children are limited, due especially to the lack of control data. In adults, gamma/delta T cells (TCR-γδ) residing in the intrathecal space are sometimes involved in neuroinflammation. To evaluate the possible role of γδ T cells in paediatric neuroinflammation, we immunophenotyped cerebrospinal fluid (CSF) and blood lymphocytes using flow cytometry in a case-control study of 100 children with non-inflammatory neurological disorders (NIND), 312 with opsoclonus-myoclonus (OMS) and 23 with other inflammatory neurological disorders (OIND). In NIND, the negative correlation between CSF γδ T cell frequency and patient age was striking: median frequency of 27% in infants and 3·3% in teens. Interindividual variations were largest in the youngest. There was no gender effect. In all OMS, after correcting for age, only a small effect of OMS severity remained. Measurement of markers for γδ T cell activation [human leucocyte antigen D-related (HLA-DR)], maturation (CD45RA, CD45RO) or intracellular cytokine staining [interleukin (IL)-4, interferon (IFN)-γ] failed to discriminate OMS and NIND groups. Of seven OMS immunotherapies/combinations, none altered the frequency of total CSF γδ T cells or subsets significantly. In OIND, the CSF γδ T cell frequency was < 10% for single samples of other paraneoplastic disorders [anti-neuronal nuclear antibody (ANNA)-1, PCA-1, teratoma-associated syndrome], cerebellar ataxia (post-infectious, ataxia-telangiectasia), acute disseminated encephalomyelitis, neuroborreliosis and encephalitis. This study provides new insights into CSF γδ T cells in the paediatric population. Although their role in CSF remains elusive, the negative age correlation, resistance to immunotherapy and our age cut-off references for NIND are important findings for the design of future paediatric studies.

6-Mercaptopurine modifies cerebrospinal fluid T cell abnormalities in paediatric opsoclonus-myoclonus as steroid sparer.

The purpose of this study was to evaluate the capacity of 6-mercaptopurine (6-MP), a known immunosuppressant, to normalize cerebrospinal fluid (CSF) lymphocyte frequencies in opsoclonus-myoclonus syndrome (OMS) and function as a steroid sparer. CSF and blood lymphocytes were immunophenotyped in 11 children with OMS (without CSF B cell expansion) using a comprehensive panel of cell surface adhesion, activation and maturation markers by flow cytometry, and referenced to 18 paediatric controls. Drug metabolites, lymphocyte counts and liver function tests were used clinically to monitoring therapeutic range and toxicity. In CSF, adjunctive oral 6-MP was associated with a 21% increase in the low percentage of CD4+ T cells in OMS, restoring the CD4/CD8 ratio. The percentage of CD4+ T cells that were interferon (IFN)-γ+ was reduced by 66%, shifting the cytokine balance away from T helper type 1 (Th1) (proinflammatory) predominance. The percentage of natural killer (NK) cells decreased significantly in CSF (-32%) and blood (-67 to -82%). Low blood absolute lymphocyte count was more predictive of improvement in CSF lymphocyte proportions (correlated with % CD4+ T cells) than the 6-thioguanine level (no correlation). 6-MP was difficult to titrate: 50% achieved the target absolute lymphocyte count (< 1·5 K/mm); 20%, the 'therapeutic' 6-thioguanine level; and 40% the non-toxic 6-methylmercaptopurine level. Side effects and transaminase elevation were mild and reversible. Clinical steroid-sparing properties and lowered relapse frequency were demonstrated. 6-MP displayed unique pharmacodynamic properties that may be useful in OMS and other autoimmune disorders. Its steroid sparer capacity is limited to children in whom the therapeutic window can be reached without limiting pharmacokinetic factors or side effects.

Expression of CXCR3 and its ligands CXCL9, -10 and -11 in paediatric opsoclonus-myoclonus syndrome.

Opsoclonus-myoclonus syndrome (OMS) is a neuroinflammatory disorder associated with remote cancer. To understand more clearly the role of inflammatory mediators, the concentration of CXCR3 ligands CXCL10, CXCL9 and CXCL11 was measured in 245 children with OMS and 81 paediatric controls using enzyme-linked immunosorbent assay (ELISA), and CXCR3 expression on CD4(+) T cells was measured by flow cytometry. Mean cerebrospinal fluid (CSF) CXCL10 was 2·7-fold higher in untreated OMS than controls. Intrathecal production was demonstrated by significantly different CXCL10 CSF : serum ratios. The dichotomized 'high' CSF CXCL10 group had higher CSF leucocyte count (P = 0·0007) and B cell activating factor (BAFF) and CXCL13 concentrations (P < 0·0001). CSF CXCL10 did not correlate with clinical severity or relapse using grouped data, although it did in some patients. Among seven types of immunotherapy, including rituximab or chemotherapy, only adrenocorticotrophic hormone (ACTH) monotherapy showed reduced CSF CXCL10, but prospective longitudinal studies of ACTH combination therapies indicated no reduction in CXCL10 despite clinical improvement (P < 0·0001). CXCL10 concentrations were 11-fold higher in CSF and twofold higher in serum by multiplexed fluorescent bead-based immunoassay than enzyme-linked immunosorbent assay, but the two correlated (r = 0·7 and 0·83). In serum, no group differences for CXCL9 or CXCL11 were found. CXCR3 expression on CD4(+) T cells was fivefold higher in those from CSF than blood, but was not increased in OMS or altered by conventional immunotherapy. These data suggest alternative roles for CXCL10 in OMS. Over-expression of CXCL10 was not reduced by clinical immunotherapies as a whole, indicating the need for better therapeutic approaches.

Unstable insertion in the 5' flanking region of the cystatin B gene is the most common mutation in progressive myoclonus epilepsy type 1, EPM1.

Progressive myoclonus epilepsy type 1 (EPM1, also known as Unverricht-Lundborg disease) is an autosomal recessive disorder characterized by progressively worsening myoclonic jerks, frequent generalized tonic-clonic seizures, and a slowly progressive decline in cognition. Recently, two mutations in the cystatin B gene (also known as stefin B, STFB) mapping to 21q22.3 have been implicated in the EPM1 phenotype: a G-->C substitution in the last nucleotide of intron 1 that was predicted to cause a splicing defect in one family, and a C-->T substitution that would change an Arg codon (CGA) to a stop codon (TGA) at amino acid position 68, resulting in a truncated cystatin B protein in two other families. A fourth family showed undetectable amounts of STFB mRNA by northern blot analysis in an affected individual. We present haplotype and mutational analyses of our collection of 20 unrelated EPM1 patients and families from different ethnic groups. We identify four different mutations, the most common of which consists of an unstable approximately 600-900 bp insertion which is resistant to PCR amplification. This insertion maps to a 12-bp polymorphic tandem repeat located in the 5' flanking region of the STFB gene, in the region of the promoter. The size of the insertion varies between different EPM1 chromosomes sharing a common haplotype and a common origin, suggesting some level of meiotic instability over the course of many generations. This dynamic mutation, which appears distinct from conventional trinucleotide repeat expansions, may arise via a novel mechanism related to the instability of tandemly repeated sequences.

Serotonin and human myoclonus. Rationale for the use of serotonin receptor agonists and antagonists.

This is a critical review of the serotonin hypothesis of myoclonus for the purpose of identifying new pharmacologic therapies. The literature on myoclonus and serotonin neuropharmacology reveals evidence for serotonergic abnormalities in some human myoclonic disorders, new serotonin receptor subtypes and data on their molecular structure and function, more selective drugs, and experimental evidence linking certain serotonin receptor subtypes with myoclonus. This article provides an overview of clinical experience with serotonergic drugs, new investigational drugs, and strategies for gathering data critical to linking particular receptor abnormalities and drugs with specific human myoclonic disorders. Such information will allow the use of receptor subtype-selective agonists and antagonists for the treatment of myoclonus.

The metabolic consequences of experimental intraventricular hemorrhage.

Experimental intraventricular hemorrhage was produced by injection of autologous fresh blood (0.25 ml/kg) or artificial CSF into the right lateral ventricle of 24 dogs. A transient ventricular fluid acidosis (pH drop to 7.09) was accompanied by increased lactate, pyruvate, ammonia, and Pco2, and decreased bicarbonate and glucose. High lactate/pyruvate ratios were the most persistent abnormality. The control group, which received intraventricular artificial CSF, developed minimal ventricular fluid acidosis (pH 7.26). Lumbar CSF and venous blood acid-base parameters did not change. Simultaneous cisternal samples obtained from some of the animals reflected similar metabolic abnormalities of lesser magnitude. Intraventricular injection of sodium bicarbonate normalized the pH in four animals.

Cortical tremor. A common manifestation of cortical myoclonus.

Ten patients, three with postural tremor and seven with action myoclonus, had stereotyped involuntary rhythmic movements when attempting to execute a sustained isometric muscle contraction. The movements were characterized by rhythmic EMG bursts lasting less than 50 msec and appearing synchronously in agonist and antagonist muscles at a rate of 9 to 18 Hz. Backaveraging of the EEG activity related to the onset of the rhythmic EMG bursts identified a cortical potential preceding the EMG bursts in all patients. These symptoms and signs fit the description of "cortical tremor," a variant of cortical reflex myoclonus. Cortical tremor is common in patients with cortical myoclonus and may be a source of functional disability. In two patients in whom we studied the effects of graded levels of isometric force, force recruitment modulated the abnormal EMG bursting frequency, amplitude, and spatial distribution of the myoclonic jerks in the activated limb. Transcranial magnetic and electrical stimulation, but not peripheral nerve stimulation, influenced the abnormal EMG bursting pattern, implying a greater dependence of this rhythmic phenomenon on a central generator than on peripheral feedback loops.

Drug-induced regulation of [125I]iodocyanopindolol-labeled 5-hydroxytryptamine1B receptor binding sites in the central nervous system.

Little is known about the regulation of 5-hydroxytryptamine1B (5-HT1B) receptors, a putative terminal autoreceptor in the central nervous system. We studied the regional responses of [125I]iodocyanopindolol ([125I]ICYP)-labeled central 5-HT1B sites to chronic treatment with 5-HT agonists and antagonists at a dose of 10 mg/kg/d IP for 30 consecutive days in the rat. In controls, there were 3.4-fold regional differences in Bmax, with a rank order of brainstem > hippocampus > cortex, striatum > spinal cord, and Kas were slightly lower in striatum and spinal cord. RU 24969 significantly reduced Bmax 23 to 63% in cortex, hippocampus, striatum, brainstem, and spinal cord without a change in Ka except for a 1.7-fold increase in cortex and spinal cord. The putative 5-HT1B agonist (m-trifluoromethyl-phenylpiperazine (TFMPP), but not [1-(3-chlorophenyl)piperazine] (m-CPP) or the 5-HT1B antagonists pindolol or quipazine, reduced the Bmax of cortical 5-HT1B sites (-16%). Chronic treatment with the 5-HT antagonists methysergide, pindolol, propranolol, ritanserin, metergoline, or methiothepin did not significantly affect striatal Bmax or Kd compared to respective vehicles. The data demonstrate significant changes in maximum number of 5-HT1B receptors in response to chronic agonist but not antagonist treatments at the dose studied.

Antimyoclonic properties of S2 serotonin receptor antagonists in the rat.

The capacity of the putative S2 serotonin receptor antagonists, pirenperone, pipamperone, ketanserin and cinanserin, to block the myoclonic syndrome produced by 30 mg/kg of L-5-hydroxytryptophan (5-HTP) [after lesioning 5-hydroxytryptamine (serotonin, 5-HT)-containing neurons with 5,7-dihydroxytryptamine (DHT)] or 15 mg/kg of fenfluramine (FF) or p-chloroamphetamine (PCA) was tested in adult male Sprague-Dawley rats. S2 antagonists inhibited limb (arrhythmic and asynchronous) and axial (truncal) myoclonus in a dose-dependent manner in the rank order of potency: pirenperone greater than pipamperone greater than ketanserin = cinanserin. Abnormal movements (myoclonus, lateral head weaving) of the myoclonic syndromes were better antagonized than postural abnormalities (hindlimb abduction, hunching of back). Centrally acting drugs, selective for S2 receptors (pirenperone, pipamperone), exhibited greater antimyoclonic properties than the non-selective 5-HT antagonist methysergide, which was as effective as ketanserin and cinanserin. Significant non-specific reduction in myoclonus without the improvement of other behavioral responses followed treatment with sedative/neuroleptic drugs, such as haloperidol (but not the non-neuroleptic dopamine antagonist sulpiride), clonazepam and diazepam. The anticonvulsants valproic acid (100 and 300 mg/kg), adrenocorticotrophic hormone (ACTH; 100 and 300 U/kg), diphenylhydantoin (15 mg/kg), and phenobarbital (20 mg/kg) and drugs which do not act principally at S2 receptors were ineffective in these models. These data support the hypothesis that myoclonus in behavioral models induced by 5-HT is S2 receptor mediated. S2 antagonists could have a role in the treatment of human myoclonus.

Brainstem serotonin receptors in the guinea pig: implications for myoclonus.

The brainstem is the locus of serotonin (5-HT)-mediated myoclonus in the guinea pig, which is induced by 5-hydroxy-L-tryptophan (L-5-HTP) and indole but not piperazine 5-HT receptor agonists. As an initial step in testing the hypothesis that one 5-HT receptor subtype mediates this effect, we measured seven 5-HT receptor binding sites and the 5-HT uptake site in guinea pig brainstem and compared them to the rat. In guinea pig brainstem, the rank order of binding site density was: 5-HT transporter site >> 5-HT1D > antagonist-labeled 5-HT2 > 5-HT1A, 5-HT1C > 5-HT1E > agonist-labeled 5-HT2 binding site. There were fewer 5-HT1A and 5-HT1C binding sites and 5-HT uptake sites in guinea pig than rat brainstem, more 5-HT1D and antagonist-labeled 5-HT2 sites, but the differences were 2-fold or less. The major species difference was that 5-HT1B sites were virtually undetectable in guinea pig brainstem. Limited competition experiments with related 5-HT receptor subtype-selective agonists and antagonists suggested that the sites in guinea pig brainstem conformed to those described in the rat. 5-HT agonist and antagonist dose-threshold and dose maximum-effect data from guinea pig myoclonus in vivo were compared with receptor affinities at each receptor site in vitro from the literature. No convincing correlation between myoclonus and one particular 5-HT site was found. These data indicate the presence of a full complement of 5-HT receptor binding site subtypes in guinea pig brainstem with some species differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of 5-HT2 receptors in rat cortex. Studies with a putative selective agonist and an antagonist.

The phenylisopropylamine derivative 1-(2,5-dimethoxy-4-iodo-phenyl)-2-aminopropane (DOI) has been suggested recently as a selective serotonin2 (5-HT2) receptor agonist. Because of the potential importance of such a tool for investigations of 5-HT2 receptor regulation, receptor binding studies were performed in rats after acute and chronic treatment with DOI, the selective 5-HT2 antagonist ketanserin, or vehicle. Single injections of 5 or 10 mg/kg DOI reduced the Bmax of cortical sites labeled with [3H]1-(2,5-dimethoxy-4-bromo-phenyl)-2-aminopropane and [3H]ketanserin (9-32 or 32-46%, respectively). Chronic daily treatment with DOI (3-9 mg/kg) further down-regulated 5-HT2 sites in cortex identified with either [3H]ketanserin (-60%) or with [3H]DOB (-75%), without altering Kd values or affecting 5-HT1 sites. In vitro addition to the [3H]ketanserin or [3H]DOB binding assay of 10 nM to 1 microM DOI resulted in competitive inhibition, suggesting that down-regulation found in vivo was not secondary to residual drug. Chronic treatment with ketanserin (10 mg/kg) also down-regulated both [3H]ketanserin (-38%) and [3H]DOB (-58%) sites in cortex without charges in 5-HT1 sites. In naive cortex, competition experiments revealed a Ki (nM) for ( +/- )-DOI of 1.7 +/- 0.02 at sites labeled by [3H]DOB, and a KH and KL of 4.8 +/- 1.5 and 53 +/- 2 nM at sites labeled by [3H]ketanserin. These data indicate that in chronic treatment, DOI, like ketanserin, is highly selective for 5-HT2 vs 5-HT1 sites at behaviorally useful doses. However, a representative putative 5-HT2 selective agonist and antagonist have similar effects on 5-HT2 receptors labeled by agonist or antagonist radioligands.

Enhanced selective 5-HT depletions in the DHT rat model: denervation supersensitivity and recovery of function.

The effects of enhancing 5-HT depletion with multiple intracisternal injections of 5,7-dihydroxytryptamine (DHT) on spontaneous or L-5-hydroxytryptophan (5-HTP)-induced behaviors (videotaped) and locomotor activity (photocell recording) were studied in the adult rat. After four DHT injections, 5-HT content in septum/accumbens, hippocampus, striatum, neocortex, cerebellum, and cervical spinal cord fell to 0-10% of controls. Multiple injections also significantly improved depletions in brainstem and diencephalon, which were not as extensive. Spontaneous locomotor activity (LMA) was increased in DHT-lesioned rats for 1 week. The associated behavioral abnormalities, hindlimb hyperextension and incomplete rearing were also transient and differed from the motor syndrome evoked by 5-HTP. Multiple DHT injections did not qualitatively modify the 5-HTP syndrome but shifted the dose response curve to the left compared to single injections. Syndrome behaviors shared a similar dose threshold and could be evoked with 30 mg/kg 5-HTP. Two weeks after DHT, the locomotor response to 5-HTP (65 mg/kg) was method dependent or biphasic: decreased in brief recordings when syndrome abnormalities were greatest and increased in hour-long recordings. LMA correlated with rearing in controls and inversely with total behavioral abnormality in DHT-lesioned rats injected with 5-HTP. Multiple regression of LMA with regional 5-HT content was significant for hippocampus, striatum, and septum/accumbens. These data suggest that the development of denervation supersensitivity, the proposed mechanism of the 5-HTP-evoked motor syndrome, may be responsible for the rapid recovery of function in LMA.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic ACTH treatment: influence on 5-HT2 receptors and behavioral supersensitivity induced by 5,7-dihydroxytryptamine lesions.

The capacity of the serotonin (5-HT) precursor 5-HIP to induce the ACTH-responsive myoclonic-convulsive disorder infantile spasms in patients with Down's syndrome has been cited as evidence for altered serotonergic neurotransmission in infantile spasms. Since there is no animal model of infantile spasms, the suitability of behavioral supersensitivity (myoclonus) evoked by 5-HTP in rats with 5,7-dihydroxytryptamine (DHT) lesions as a model was tested by determining the effect of chronic treatment with ACTH (40 IU/kg) on 5-HTP-evoked myoclonus. In rats treated with DHT as adults, ACTH administration did not alter the "serotonergic behaviors," such as myoclonus, induced by 30 mg/kg 5-hydroxytryptophan (5-HTP), but induced a small significant increase in Bmax of neocortical 5-HT2 sites of the DHT group, with no change in rats without lesions. In rats treated with DHT as neonates, there was also no significant difference in behaviors evoked by several doses of 5-HTP. These data suggest that ACTH minimally modifies the effects on 5-HT receptors of DHT lesions, but the intracisternal DHT model is not a suitable model for infantile spasms because chronic ACTH was not antimyoclonic.

High-dose clonidine motor syndrome: relationship to serotonin syndrome.

Reciprocal forepaw treading, hindlimb abduction, and Straub tail are some of the abnormal motor behaviors of the classical 'serotonin syndrome,' which results from activation of serotonin (5-HT) receptors. However, we also observed them in the syndrome evoked by the alpha-adrenergic agonist clonidine, at high doses (5-40 mg/kg). Other features of the clonidine syndrome (scored from videotapes) were body and head tremor, forelimb hyperextension, ataxia, vertical jumping, tactile hyperreactivity, and autonomic signs (piloerection, pupillary dilatation, salivation, proptosis). The clonidine syndrome persisted for several hours and was not lethal. Clonidine suppressed locomotor activity (photocell recording) and induced episodes of catalepsy and 5-HT-independent impairment of motor habituation. Single high doses of drugs active at several different neurotransmitter receptors significantly reduced total behavioral score through effects primarily on tremor and autonomic signs, but none prevented the clonidine syndrome. Lesions of monoaminergic neurons [intracisternal 5,7-dihydroxytryptamine (DHT) or 6-hydroxydopamine] or monoamine depletion by intraperitoneal reserpine all failed to prevent this motor syndrome. Co-administration of 5-HTP and clonidine did not exacerbate the clonidine syndrome in naive rats and did not prevent the onset of the serotonergic syndrome in rats with DHT lesions. These data suggest that neither catecholamines nor 5-HT have a major role in the serotonin-like behavioral responses to high doses of clonidine.

Serotonin-lesion myoclonic syndromes. II. Analysis of individual syndrome elements, locomotor activity and behavioral correlations.

This study evaluated the behavioral elements of three 5-HT-related syndromes (intraperitoneal 5-hydroxytryptophan after intracisternal 5,7-dihydroxytryptamine (DHT), p-chloroamphetamine (PCA), fenfluramine (FF), or combinations of drugs) scored from video-tapes and their relationship to locomotor activity (LMA) photocell recording, regional monoamine concentration and S-1 receptor binding. Rearing was eliminated by drugs which produce the myoclonic syndrome and was the single best indicator of control treatments (saline or 5-HTP in unlesioned rats and saline in DHT-lesioned rats). Global 'abnormality', hunching (rigid arching of back), hindlimb abduction, forepaw myoclonus, stereotyped lateral head movements, backing, and immobility occurred significantly only in drug-treated rats. Multiple forms of myoclonus (appendicular and truncal) and convulsions were dose-dependent drug effects. Both 5-HTP (after DHT) and PCA increased LMA significantly, but hyperactivity induced by PCA could be blocked by giving 5-HTP concomitantly. Substantial 5-HT presynaptic destruction by DHT prevented backing but not other behavioral or locomotor effects of FF and PCA. Drug combinations did not produce additive behavioral effects. Backing, immobility, and locomotor activity best differentiated between drug treatments, and could be used to correctly allocate animals to drug groups. Drug treatments also could be differentiated by reducing the number of behavioral variables into summary variables (principal components) and by discriminant analysis. Only forepaw myoclonus and total behavioral score were correlated with 5-HT concentrations (brainstem), indicating behavioral heterogeneity. Our study suggests that there is a common core 'myoclonic-serotonergic' syndrome (forepaw myoclonus, head weaving, hindlimb abduction, hunching) of stimulation of 5-HT receptors plus additional drug-specific elements (backing, LMA). Although brainstem receptors appear to be an important locus for some of these behaviors, S-1 receptors do not explain the behavioral supersensitivity to 5-HTP in our DHT-lesioned rats.

Serotonin-lesion myoclonic syndromes. I. Neurochemical profile and S-1 receptor binding.

This paper and the following one describe the effects of L-5-hydroxytryptophan (5-HTP) (after 3 intracisternal injections of 5,7-dihydroxytryptamine (DHT], fenfluramine (FF), p-chloroamphetamine (PCA) and drug combinations on (i) brain regional amine concentration (HPLC with LEC) and serotonin S-1 receptor binding; and (ii) 'serotonergic' behaviors in the same adult rats. Serotonin (5-HT) neurotoxins produced significantly different regional profiles of 5-HT depletion. Multiple DHT injections caused a 90-100% depletion of 5-HT concurrently in neocortex, hippocampus, striatum, septum/accumbens, pons, cerebellum, and cervical cord. Only PCA significantly depleted midbrain. Drug combinations with DHT resembled DHT alone rather than additive depletions, except for PCA + DHT, which produced a hybrid pattern of depletion. The S-1 binding assay, using cold 5-HT to displace [3H]5-HT, was performed with and without ascorbate, EDTA, CaCl2, and pargyline. Without ascorbate, binding was specific, saturable, region-dependent, and non-linear with high (Kd 1-3 nM) and low affinity (10-20 nM) components but no cooperativity (0.8 less than nH less than 1.0). Bmax and Kd did not differ significantly between vehicle- and drug-treated animals in neocortex, hippocampus, striatum, thalamus, hypothalamus, midbrain, pons, medulla, cervical cord, cerebellum, or septum/accumbens two weeks after lesioning, while the assay did detect a 60% reduction in Bmax induced by ascorbic acid (1 mM). The effects of assay conditions exceeded the changes sometimes reported in S-1 receptor Bmax after 5-HT lesions.

Plastic responses of neonatal 5-hydroxytryptamine1B receptors to 5,7-dihydroxytryptamine lesions mapped by quantitative autoradiography.

We previously found different effects on behavior, serotonin (5-HT) concentrations, 5-HT uptake sites, and 5-HT1A binding sites of neonatal 5,7-dihydroxytryptamine (5,7-DHT) lesions depending on the route of 5,7-DHT injection. To study the impact of early lesions on 5-HT1B sites as putative 5-HT terminal autoreceptors, we labelled them autoradiographically with [3H]5-HT 4 months after intraperitoneal (i.p.) or intracisternal (i.c.) 5,7-DHT injection during the first postnatal week and quantitated specific binding in 22 brain regions. Changes were confined to the subiculum and substantia nigra, regions with the most 5-HT1B-specific binding and projection areas of structures with high mRNA expression. Both routes of 5,7-DHT injection were associated with increases in specific binding in subiculum (24% for i.p. and 47% for i.c. route). In contrast, there was a 32% increase in specific binding in the substantia nigra in rats with lesions made i.c. but not i.p. No significant differences were found in nucleus accumbens, caudate-putamen or other brain areas. In saturation homogenate binding studies of 5-HT1B sites using [125I]iodocyanopindolol 1 month after i.p. injections, neonatal 5,7-DHT lesions did not significantly alter Bmax or Kd in the neocortex, striatum, diencephalon or brainstem. These data indicate the differential effects of the route of neonatal 5,7-DHT injections on plasticity of 5-HT1B receptor recognition sites and suggest the presence of a subpopulation of post-synaptically located 5-HT1B sites which increases in response to denervation. The data also suggest that sprouting of 5-HT neurons after neonatal 5,7-DHT lesions does not involve 5-HT1B sites.

The guinea pig myoclonic model: behavioral supersensitivity to 5-hydroxytryptophan induced by intracisternal 5,7-dihydroxytryptamine.

To study the species difference of guinea pigs and rats in response to 5-hydroxytryptophan (5-HTP), we injected both animals intracisternally with 5,7-dihydroxytryptamine. In rats with 5,7-dihydroxytryptamine lesions, 5-HTP evoked the well described myoclonic-serotonergic syndrome. In the guinea pig, 5,7-dihydroxytryptamine lesions significantly increased the severity of myoclonic response to 5-HTP (150 mg/kg) compared to vehicle controls, resulting in lethal convulsions. Guinea pigs treated with 5,7-dihydroxytryptamine did not develop spontaneous myoclonus, or when treated with 5-HTP, other 'serotonergic behaviors' such as lateral head weaving, hindlimb abduction, and forepaw tapping. Guinea pigs tolerated intracisternal 5,7-dihydroxytryptamine less well than rats, with a higher mortality, although immediate post-injection convulsions were less severe and did not require phenobarbital prophylaxis. Staged lower doses of 5,7-dihydroxytryptamine (100-200 micrograms) were better tolerated than a single high dose of neurotoxin (400 micrograms). The regional profile of 5,7-dihydroxytryptamine lesions in the guinea pig resembled that of the rat, with maximal depletion of 5-HT in spinal cord and selected forebrain structures, and little effect in diencephalon and midbrain. Depletions in the guinea pig were less selective for 5-HT using desipramine pretreatment than in the rat. In naive guinea pigs and rats, regional content of 5-HT was similar. These data suggest that the functional integrity of serotonergic neurons is not requisite for the expression of myoclonus induced by 5-HTP in the guinea pig. 5,7-Dihydroxytryptamine lesions in the guinea pig resulted in behavioral and neurochemical similarities and differences in comparison with the rat.

ACTH binds to [3H]MK-801-labelled rat hippocampal NMDA receptors.

The molecular mechanism of the clinical antiepileptic/antimyoclonic action of adrenocorticotrophic hormone (ACTH) is unknown. To explore the possible role of excitatory amino acid receptors, we studied the influence of ACTH and ACTH fragments in vitro on the binding of [3H]MK-801 to rat hippocampus, a region relevant to epilepsy. ACTH-(1-39), ACTH-(1-24), and ACTH-(1-17) displayed micromolar affinities compared to the nanomolar affinity of MK-801, whereas ACTH-(4-10) and four clinically used anticonvulsants were inactive. These findings are not specific for NMDA receptors but conform to the rank order of potency of ACTH fragments at other receptor binding sites.

Novel regulation of 5-HT1C receptors: down-regulation induced both by 5-HT1C/2 receptor agonists and antagonists.

The 5-hydroxytryptamine1C (5-HT1C) receptor shares many features with the 5-HT2 receptor. To determine if the regulation of the sites is also similar we studied the effects of chronic treatment with drugs active at 5-HT1C/2 receptors on [3H]mesulergine-labelled 5-HT1C binding sites in spinal cord. The 5-HT receptor agonists 1-(3-chlorophenyl)piperazine (m-CPP) (-38%), 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (-35%), quipazine (-27%) and m-trifluoromethylphenylpiperazine (TFMPP) (-27%) significantly down-regulated spinal 5-HT1C sites with chronic injection compared to vehicle treatment. The 5-HT receptor antagonists methiothepin (-71%), mianserin (-24%), methysergide (-21%), and cyproheptadine (-27%) also induced down-regulation, and ritanserin and metergoline further reduced [3H]mesulergine specific binding to undetectable levels. There were no significant changes in Kd to implicate presence of residual drug except for mianserin, methiothepin, and TFMPP. Pindolol and spiperone had no significant effects. In acute dose-response studies, injection of a single dose of DOI did not result in a significant change in any receptor parameters. The capacity of a drug to lower Bmax correlated significantly with its pKd (r = 0.84, P < 0.0007). This drug regulation pattern for 5-HT1C sites of down-regulation by both 5-HT1C/2 receptor agonists and antagonists is similar to that for 5-HT2 receptors and is consistent with the classification of 5-HT1C and 5-HT2 receptors in the same superfamily.