Neurodegenerative Effects Induced by Chronic Infusion of Quinolinic Acid in Rat Striatum and Hippocampus.

In this study we examined whether the potency of quinolinic acid (Quin) in inducing neurodegeneration in vivo was dependent on the exposure time of the tissue to the excitotoxin. The effect of chronic infusion of Quin into rat striatum and hippocampus was examined at the light microscopic level and by cell count on 40 microm Cresyl violet stained brain sections. Continuous infusion was at a constant speed (0.5 microl/h) for various times (15 h - 2 weeks) by osmotic minipumps (Alzet 2002). No build up of [3H]Quin occurred in the tissue during infusion; this was assessed by measuring the radioactivity 3 - 14 days after minipump placement. Intrastriatal infusion of 6 and 10 nmol/h Quin, but not of nicotinic acid, for 1 week induced a dose-dependent neurodegeneration (70 and 90% loss of neurons, respectively, compared to the contralateral striatum) extending 1.2 - 2 mm from the centre of the injection. The onset of the neurotoxicity caused by 10 nmol/h Quin was >24 h. One week's infusion of 4 nmol/h Quin did not induce neurotoxicity, but a 40% drop of neurons, compared to the contralateral side, occurred after 2 weeks. One week's intrahippocampal infusion of 2.4 and 6 nmol/h Quin, but not of nicotinic acid, caused a dose-dependent neurodegeneration with a radius of approximately 1 - 1.5 mm around the injection track. The onset of the neurotoxicity induced by 2.4 nmol/h Quin was < 15 h. The pattern of nerve cell loss induced by 1.2 nmol/h Quin after 1 week (CA4 cells lost in 50% of the rats) did not differ from that observed after 2 weeks of infusion. Nerve cell loss caused by Quin in the striatum and in the hippocampus was restricted to the injected area and antagonized by coinfusion with d(-)-2-amino-7-phosphonoheptanoic and kynurenic acids in molar ratios of 1:0.1 and 1:3, respectively. These data show that Quin's potency in inducing neurodegeneration in the striatum, but not in the hippocampus, depends on the exposure time of the tissue to the excitotoxin. In addition, neurodegeneration is induced faster by Quin in the hippocampus than in the striatum. The usefulness of this model to study the sequelae of the neurotoxic process in vivo will be discussed.

Distribution of GAP-43 mRNA in the adult rat brain.

Regional distribution of gene expression of the axonal growth-associated protein, GAP-43, was studied in adult rat brains by in situ hybridization autoradiography to determine the features of mature neuronal populations that synthesize GAP-43 protein. Such synthesis appears to correlate with axonal growth during maturation and regrowth after axotomy. In most adult neurons, the sharp decline in GAP-43 gene expression implies a reduced capacity for axonal growth. Neurons capable of extending axonal knobs in the absence of injury may indicate a "plasticity" underlying dynamic processes of interaction between neurons and their synaptic targets. Antisense and sense (control) riboprobes were used on serial sections in the three principal axes, and the magnitude of hybridization signal was examined to determine regional patterns. GAP-43 mRNA levels are pronounced in diverse neuronal groups including the locus coeruleus, raphé nn., dopaminergic nigral and ventral tegmental nn., mitral cells, hippocampal CA3, inferior olivary n., vagal motor n. and other parasympathetic preganglionic neurons, select thalamic midline and intralaminar nn., several specific nn. of the hypothalamus and basal forebrain, the granular layer of cerebellar cortex, the infragranular neocortex, and the granular olfactory paleocortex; there is a substantial range in the magnitude of expression. Regions revealing minimal signal include most thalamic sensory relay nuclei, the granule neurons of the olfactory bulb and dentate gyrus, and the caudate and putamen. Possible concomitants of GAP-43 expression include regulation of ion flux and neurotransmitter release. Those neurons with long, extensively dispersed and numerous synaptic connections display the strongest signals and may possess the greatest propensity for continuous growth and turnover of their axon terminals, in contrast to short-axon and specific projection neurons exhibiting minimal levels. These data may enable inferring which populations display normal or experimentally induced axonal growth.

Evidence that dopamine mechanisms in the nucleus accumbens are selectively involved in the effect of desipramine in the forced swimming test.

Bilateral injections of 5 or 1 (but not 0.5) micrograms sulpiride into the nucleus accumbens reduced the effect of a 7-day treatment with 10 mg/kg/day desipramine in the forced swimming test. Bilateral injections of 5 or 1 micrograms sulpiride in the caudate-putamen did not modify the anti-immobility effect of desipramine. The data support the hypothesis that dopamine mechanisms in the limbic regions of the brain of the rat are involved in the effect of repeated treatment with desipramine in the forced swimming test.

Role of serotonin and catecholamines in brain in the feeding suppressant effect of fluoxetine.

The hypophagic effect of fluoxetine was studied in rats, injected intracerebroventricularly with 150 micrograms/20 microliters 5,7-dihydroxytryptamine, to destroy serotonin-containing neurones or 250 micrograms/20 microliters 6-hydroxydopamine, to destroy catecholamine-containing neurones. The effect of various serotonin receptor antagonists was assessed as well. Neither neurotoxin significantly modified the effect of 20 mg/kg (i.p.) fluoxetine on food intake. Metergoline (1-5 mg/kg), (-)-propranolol (16 mg/kg) and ICS 205-930 (0.1 and 1 mg/kg) did not modify the hypophagic effect of fluoxetine, while mianserin (1 and 5 mg/kg), ritanserin (0.5 and 1 mg/kg) and xylamidine (3 mg/kg) slightly but significantly reduced it. While the mechanism by which some 5-HT receptor antagonists modify the effect of fluoxetine remains to be elucidated, it seems clear that 5-HT receptors hardly have any significant role in the ability of the drug to suppress food intake.

Release of dopamine is reduced by diazepam more in the nucleus accumbens than in the caudate nucleus of conscious rats.

The effects of 1-20 mg/kg diazepam were studied on the extracellular concentrations of dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the nucleus accumbens and striatum of conscious rats, using intracerebral microdialysis. Five, but not 1 mg/kg diazepam significantly reduced extracellular DA, DOPAC and HVA in the nucleus accumbens. Twenty mg/kg diazepam significantly reduced extracellular DA, DOPAC and HVA in the striatum. A significant effect on striatal DOPAC, but not on DA and HVA, was seen with 10 mg/kg diazepam, while no changes were found with 5 mg/kg diazepam. The results suggest that diazepam reduces the release and metabolism of DA in the nucleus accumbens more than in the striatum.

The role of serotonin and dopamine in brain in the antidepressant-like effect of clonidine in the forced swimming test.

The effect of clonidine (0.1 mg/kg, i.p.), as a three-injection course, on behaviour in the forced swimming test was studied in rats injected intracerebroventricularly (i.c.v.) with 150 micrograms 5,7-dihydroxy-tryptamine (5,7-DHT) to destroy serotonin (5-HT) neurones or treated with 100 mg/kg (i.p.) (+/-)-sulpiride or 0.5 micrograms/0.5 microliter (-)-sulpiride in the nucleus accumbens. Clonidine significantly increased struggling and reduced floating and the effects were antagonized by both treatments with sulpiride but not by 5,7-DHT which markedly depleted 5-HT in brain. The results suggest that the mesolimbic dopaminergic system but not 5-HT neurones, plays a permissive role in the antidepressant-like effect of clonidine in the forced swimming test.

Selective reduction of extracellular dopamine in the rat nucleus accumbens following chronic treatment with DAU 6215, a 5-HT3 receptor antagonist.

The effect of chronic treatment with (3-alpha-tropanyl)1H-benzimidazolone-3-carboxamide chloride (DAU 6215; 15 micrograms/kg s.c. twice daily for 21 days), a serotonin3 receptor antagonist, on the extracellular concentrations of dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was studied by intracerebral dialysis in the striatum, nucleus accumbens and frontal cortex of conscious rats. Twenty-four hours after the last injection, the basal extracellular concentrations of DA in the nucleus accumbens of rats given DAU 6215 were significantly lower than in saline-treated rats. DA output in the dorsolateral striatum or frontal cortex was not significantly different between the DAU 6215 and saline-treated rats. Chronic DAU 6215 significantly reduced the extracellular concentrations of DOPAC and HVA in the frontal cortex but had no effect in the other brain regions. A subcutaneous challenge dose of DAU 6215 (15 micrograms/kg) did not significantly modify the extracellular concentrations of DA and its metabolites in either DAU 6215 or saline treated rats in any of the brain regions examined. The present investigation is the first on the effect of chronic administration of a 5-HT3 receptor antagonist on basal extracellular DA in the rat brain. The results provide evidence of an association between the electrophysiological and biochemical effects of chronic treatment with a serotonin3 receptor antagonist on the activity of the mesolimbic DA system. In line with the theory that hyperactivity of the mesolimbic dopaminergic system is involved in psychosis, the results suggest that DAU 6215 may be useful in the treatment of psychotic disorders, possibly with limited extrapyramidal effects.

Effect of 5-HT1A agonists on stress-induced deficit in open field locomotor activity of rats: evidence that this model identifies anxiolytic-like activity.

Deficits in locomotion and exploratory behaviour in an open field were induced in rats by restraint for 2 hr, 23 hr before testing. Diazepam, 0.62 and 1.25 mg/kg, intraperitoneally (i.p.), 15 min before testing, reversed the stress-induced reduction in locomotion; 1.25 mg/kg also attenuated the effect of stress on exploration (rearing and object exploring). Diazepam did not affect the activity of controls. A putative anxiogenic compound, pyrazoloquinoline (CGS 8216, 10 mg/kg administered 30 min before testing), also markedly reduced locomotion and exploration and the effect was reversed by 2.5 mg/kg diazepam, 15 min before testing. Buspirone, 0.1 mg/kg subcutaneously (s.c.) 15 min before testing, significantly attenuated the effect of stress on locomotion and exploration but had no effect in controls. Larger doses (0.5 and 1.0 mg/kg) markedly reduced the behavioural measures in controls and did not modify or enhance the effect of stress. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), 0.25 and 0.5 mg/kg (s.c.), 1 hr before testing, significantly attenuated the reduction in locomotion without affecting rearing and object-exploring in stressed rats. At doses from 0.125 to 0.5 mg/kg, 8-OH-DPAT reduced exploration in control rats. Two hr after restraint (corresponding to 21 hr before testing in the open field) 8-OH-DPAT, 0.125 to 2 mg/kg (s.c.), did not modify the open field deficits, caused by stress. In these treatment conditions, 0.5 and 2 mg/kg 8-OH-DPAT reduced locomotion and exploration in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Tianeptine increases the extracellular concentrations of dopamine in the nucleus accumbens by a serotonin-independent mechanism.

The effect of various doses of tianeptine on the extracellular concentrations of dopamine was studied in the striatum and nucleus accumbens of the rat. At 5 (but not 2.5) mg/kg intraperitoneally, tianeptine increased the extracellular dopamine only in the nucleus accumbens. At 10 mg/kg, the effect was also seen in the striatum but it was less marked and shorter-lasting. At 10 mg/kg (i.p.), tianeptine significantly raised the extracellular concentrations of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in both regions. The effect of 10 mg/kg tianeptine on dopamine and its metabolites was not significantly changed in animals which had received this dose twice daily for 15 days. Intracerebroventricular administration of 150 micrograms/20 microliters 5,7-dihydroxytryptamine, which markedly depleted serotonin in the brain, did not modify the effect of 10 mg/kg tianeptine on the extracellular concentrations of dopamine and HVA in the nucleus accumbens but reduced the effect on DOPAC. Various doses of tianeptine (1, 3 and 10 mg/kg i.p.) did not change the synthesis of serotonin and dopamine in the striatum and nucleus accumbens. The results show that tianeptine increased the extracellular concentrations of dopamine more in the nucleus accumbens than in striatum. The effect on the output of DA in the nucleus accumbens could be involved in the antidepressant activity of tianeptine.

8-Hydroxy-2-(di-n-propylamino) tetralin, a selective serotonin1A receptor agonist, blocks haloperidol-induced catalepsy by an action on raphe nuclei medianus and dorsalis.

The selective serotonin1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) was studied for its ability to reverse haloperidol-induced catalepsy in rats. Given subcutaneously 8-OH-DPAT (0.06-0.5 mg/kg), dose-dependently antagonized the catalepsy induced by 1 mg/kg of haloperidol. Intraventricular injection of the serotonin (5-HT) neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), which caused marked depletion of 5-HT in brain, did not change haloperidol-induced catalepsy per se, but completely antagonized the anticataleptic effect of subcutaneously administered 8-OH-DPAT. When injected directly into the median or dorsal raphe nucleus, 8-OH-DPAT, in doses ranging from 0.2 to 5 micrograms/0.5 microliter, reduced the catalepsy induced by haloperidol. The results suggest that the activation of 5-HT1A receptors, probably those located presynaptically on 5-HT-containing cell bodies, reduces the catalepsy induced by haloperidol.

Roles of 5-HT(1A) receptors in the dorsal raphe and dorsal hippocampus in anxiety assessed by the behavioral effects of 8-OH-DPAT and S 15535 in a modified Geller-Seifter conflict model.

8-OH-DPAT [8-hydroxy-2-(di-N-propylamino)tetralin], a 5-HT(1A) receptor agonist, and S 15535 (4-benzodioxan-5-yl)1-(indan-2-yl)piperazine, a partial agonist at 5-HT(1A) receptors, were administered into the dorsal raphe nucleus and dorsal hippocampus and their behavioral effects were assessed in a modified Geller-Seifter conflict model. Injected into the dorsal raphe nucleus 8-OH-DPAT, 1 microg but not 0.04 or 0.2 microg 0.5 microl(-1), and S 15535, 2.5 microg but not 0.1 or 0.5 microg 0.5 microl(-1), significantly increased punished responding with no effect on rates of unpunished or time-out responding. WAY 100635, a selective 5-HT(1A) receptor antagonist, injected subcutaneously at 0. 3 mg kg(-1) 30 min before 1 microg 8-OH-DPAT or 2.5 microg S 15535 in the dorsal raphe, completely antagonized their effects on punished responding. At doses ranging from 1 to 10 microg microl(-1) injected into the CA1 region of the dorsal hippocampus neither 8-OH-DPAT nor S 15535 modified punished responding or the rates of time-out. At the highest doses, 8-OH-DPAT significantly reduced unpunished responding whereas S 15535 had the opposite effect. The results suggest that stimulation of 5-HT(1A) receptors in the dorsal raphe nucleus has anxiolytic-like effects whereas stimulation of postsynaptic receptors in the dorsal hippocampus has no anxiolytic or anxiogenic effects, at least judging from changes in rates of punished responding. These results are compatible with the hypothesis that 5-HT(1A) receptor agonists and partial agonists attenuate anxiety by reducing serotonergic transmission in brain areas innervated by the dorsal raphe nucleus.

WAY 100635, a 5-HT1A receptor antagonist, prevents the impairment of spatial learning caused by blockade of hippocampal NMDA receptors.

We studied the effect of WAY 100635, a 5-HT1A receptor antagonist, on the impairment of spatial learning caused by intrahippocampal injection of 3-((R)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), a competitive NMDA receptor antagonist, in a two-platform spatial discrimination task. CPP, 3 and 10 ng/microl, administered bilaterally into the CA1 region of the dorsal hippocampus 10 min before each training session, dose-dependently reduced choice accuracy in the two-platform spatial discrimination task with little or no effect on choice latency and errors of omission. A volume of 10 ng/microl intrahippocampal CPP did not affect choice accuracy or latency of a non-spatial visual discrimination task. Subcutaneous doses of 0.3 and 1 mg/kg WAY 100635 did not modify the choice accuracy, but prevented the impairment caused by 10 ng/microl intrahippocampal CPP. A dose of 20 ng/microl WAY 100635 into the dorsal hippocampus prevented the deficit caused by 10 ng/microl CPP administered in the same region. The results suggest that blockade of 5-HT1A receptors can compensate the loss of NMDA-mediated excitatory input to pyramidal cells in the hippocampus. These findings may have clinical relevance for the symptomatic treatment of memory disorders associated with reduced glutamate transmission mediated by NMDA receptors.

Effects of intracerebroventricular administration of d-fenfluramine and d-norfenfluramine, as a single injection or 2-hr infusion, on serotonin in brain: relationship to concentrations of drugs in brain.

The effects of d-fenfluramine (DF) and d-norfenfluramine (DNF), administered intracerebroventricularly (i.c.v.) on levels of serotonin (5-HT) in the brain, was assessed in relation to levels of drugs in brain. d-Fenfluramine, as a single injection (500 micrograms/20 microliters), caused no significant changes in 5-HT in whole brain from 15 to 480 min after injection. When infused intraventricularly for 2 hr, DF and DNF at 500 but not at 125 250 micrograms/hr, markedly reduced concentrations of 5-HT in brain 4 hr after the end of the infusion. At this time levels of DNF in brain were similar (between 4 and 5 micrograms/g) with both compounds, whereas levels of DNF after single intraventricular injections of DF were below 2 micrograms/g at all times after injection. Infusion of 500 micrograms/hr of DNF for 2-hr reduced concentrations of 5-HT in various regions of the brain, with the exception of the brainstem, whereas 250 micrograms/hr of DNF significantly lowered levels of 5-HT only in the cortex. The effect of infusion of 500 micrograms/hr of DNF was specific for 5-HT (no effect on dopamine and norepinephrine) and lasted for at least 168 hr. The results suggest that the effect on 5-HT in brain of intraventricular infusion of DF, but not a single injection, was due to the fact that, only in the former condition were adequate levels of DNF, the active metabolite of DF, reached in the brain. These results are relevant to the interpretation of studies in which biochemical changes in the brain after intraventricular administration, are reported without any measurement of the drug or its active metabolites, in plasma and brain.

Effect of 5-HT1A receptor antagonists on citalopram-induced increase in extracellular serotonin in the frontal cortex, striatum and dorsal hippocampus.

The aim of the present study was to compare the effects of citalopram, either alone or combined with 5-HT1A receptor antagonists, on extracellular serotonin levels in brain regions innervated by the dorsal or median raphe nuclei. Using intracerebral microdialysis in awake rats with separate probes in the frontal cortex or dorsal hippocampus, we studied the ability of 8 mg/kg s.c. (-)penbutolol, a beta-adrenoceptor antagonist with antagonist action at 5-HT1A and 5-HT1B receptors, and 0.3 mg/kg s.c. WAY-100635, a selective 5-HT1A receptor blocker, to modify the effect of 1 and 10 mg/kg i.p. citalopram on extracellular serotonin. Both doses of citalopram had more effect on extracellular serotonin levels in the dorsal hippocampus than in the frontal cortex. The effect of 1 mg/kg citalopram was significantly potentiated by (-)penbutolol in the frontal cortex only, but a clear-cut potentiation of the effect of citalopram was seen in both regions at a dose of 10 mg/kg. The effect of 10 mg/kg citalopram was potentiated by WAY-100635 in the frontal cortex but not in the dorsal hippocampus. In a second set of experiments, the combined effect of WAY-100635 and citalopram was studied in the same rat implanted with vertical probes in the striatum and dorsal hippocampus. Citalopram (1 and 10 mg/kg i.p.) raised extracellular serotonin to a similar extent in both regions. However, 0.3 mg/kg s.c. WAY-100635 potentiated the effect of 10 mg/kg citalopram in the striatum but not in the dorsal hippocampus. The results suggest that only a combined blockade of 5-HT1A and 5-HT1B receptors potentiates the effect of citalopram on extracellular concentrations of serotonin in the dorsal hippocampus. The findings may be relevant in designing clinical trials aimed at enhancing the antidepressant action of selective serotonin re-uptake inhibitors by combining them with serotonin receptor antagonists.

A peptidase-resistant cyclic octapeptide analogue of somatostatin (SMS 201-995) modulates seizures induced by quinolinic and kainic acids differently in the rat hippocampus.

Electroencephalographic (EEG) seizures were measured in rats after intrahippocampal injection of 120 nmol quinolinic acid into the stratum radiatum CA1 or 0.19 nmol kainic acid in the dentate gyrus or in the stratum radiatum. Injection of 5 micrograms SMS 201-995, a peptidase-resistant cyclic octapeptide analogue of somatostatin, into the stratum radiatum, 15 min before quinolinic acid, did not significantly modify the number of seizures and the total time in seizures. Five micrograms SMS 201-995 injected into the stratum radiatum reduced the number of seizures induced by kainic acid in the same area and the total time spent in seizures by 58% and 75%, respectively (Student's t-test; P less than 0.01). In both instances the latency to the first ictal episode was not significantly modified. Lesions of the medial septum, which reduced the activity of choline-o-acetyl-transferase (CAT) in the dorsal hippocampus by greater than 90% after one week did not significantly affect seizures induced by quinolinic acid. In rats lesioned in the medial septum, 5 micrograms SMS 201-995 reduced the total time spent in seizures by 43%, without changing the number of ictal episodes and raised the latency to the first quinolinic acid-induced seizure by 53% (ANOVA 2 x 2, P less than 0.05) but had no effect on these measures in the corresponding sham-operated group.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of various calcium channel blockers on three different models of limbic seizures in rats.

Voltage-dependent calcium channel-blockers were studied for their ability to modulate limbic seizures induced in rats by injection of quinolinic acid and kainic acid into the hippocampus or by hippocampal kindling. Flunarizine, at 40 mg/kg (but not 20 mg/kg), reduced the total number of seizures and total time spent in seizures induced by quinolinic acid by 75%; at 60 mg/kg, both parameters were reduced more than 90%, while at 80 mg/kg seizures induced by kainic acid were not affected. Forty and 60 mg/kg of flunarizine protected hippocampal-kindled rats from fully developed convulsions (Stage 5). Nifedipine, at 20 and 40 mg/kg, was ineffective on seizures induced by both quinolinate and kainate. However, at 20 mg/kg, 57% of the kindled animals were protected from Stage 5 and total protection was achieved at 40 mg/kg. Verapamil, at 40 mg/kg, reduced by respectively, 88% and 78%, the total number of seizures and the total time spent in seizures induced by quinolinic acid, but had no effect on seizures induced by kainate and Stage 5 seizures. The results suggest that, while seizures induced by kainic acid were refractory to all voltage-dependent calcium channel blockers, binding sites affected by flunarizine and verapamil in the brain may selectively facilitate ictal activity induced by quinolinic acid. Binding sites for dihydropyridine might contribute to the increased hippocampal excitability in kindled animals. The role of calcium entry through voltage-dependent calcium channels in the occurrence of seizures in these models of limbic epilepsy is discussed.

Evidence that central 5-HT2 receptors do not play an important role in the anorectic activity of D-fenfluramine in the rat.

To gain information on the role of central 5-HT2 receptors in the reduction of food intake caused by D-fenfluramine in rats, different intraperitoneal doses of metergoline, a non-selective 5-HT receptor antagonist and ritanserin, a selective 5-HT2 receptor antagonist, were compared for their ability (a) to antagonize the anorectic effect of D-fenfluramine; (b) to occupy central 5-HT2 receptors in vivo (measured by the binding of [3H]spiperone in the frontal cortex) and (c) to affect the concentrations of D-fenfluramine and its active metabolite, D-norfenfluramine in brain. Metergoline dose-dependently reduced the effect of D-fenfluramine (2.5 mg/kg i.p.) on food intake, with complete antagonism at 1 mg/kg, a dose which occupies about 50% of cortical 5-HT2 receptors. Ritanserin, at a dose (0.5 mg/kg) causing 50% occupation of 5-HT2 receptors, had no effect on anorexia induced by D-fenfluramine and only partially prevented it at doses which caused maximum occupation of 5-HT2 receptors (1-2 mg/kg). Unlike 1 mg/kg metergoline, 1 mg/kg ritanserin significantly reduced the concentrations of D-norfenfluramine in the frontal cortex and hypothalamus of rats 30 min after injection of D-fenfluramine. The results suggest that 5-HT receptors, other than 5-HT2, possibly 5-HT1B, are involved in the anorectic effect of D-fenfluramine in food-deprived rats.

Status of somatostatin receptor messenger RNAs and binding sites in rat brain during kindling epileptogenesis.

In situ hybridization histochemistry with somatostatin sst1-sst5 receptor messenger RNA-selective oligoprobes and quantitative receptor autoradiographic binding studies using [125I]Tyr3-octreotide, [Leu2,D-Trp22,125I-Tyr25]somatostatin-28 and [125I]CGP 23996 ([125I]c[Asn-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) were performed to determine the level of expression of somatostatin receptor messenger RNA and receptor binding sites in the hippocampal formation, limbic system and cerebral cortex of adult rats electrically kindled in the dorsal hippocampus. In control rats (implanted with electrodes but not electrically stimulated), the somatostatin-1 receptor-selective [125I]Tyr3-octreotide and the non-subtype-selective [Leu3,D-Trp22,125I-Tyr25]somatostatin-28 preferentially labelled the strata oriens and radiatum of the CA1 subfield of the hippocampus, the molecular layer of the dentate gyrus, the subiculum and presubiculum of the hippocampal formation, the inner layer of the frontal cortex, and the lateral and basolateral nuclei of the amygdala. The non-subtype-selective radioligand [125I]CGP 23996 (in 5 mM Mg2+ buffer) preferentially labelled the strata oriens and radiatum of the CA1 subfield of the hippocampus, the subiculum and the basolateral nucleus of the amygdala. Under conditions where primarily somatostatin-2 receptors were labelled, [125I]CGP 23996 (in 120 mM Na+ buffer) showed strong binding in the strata oriens and radiatum of the CA1 subfield of the hippocampus and the frontal cortex, whereas the dentate gyrus, subiculum and amygdala showed only weak signals. During and after kindling, no significant differences were observed between the ipsi- and contralateral sides of the hippocampus. A significant decrease (about 40%) of somatostatin receptor binding sites was observed in the molecular layer of the dentate gyrus with all radioligands (except [125I]CGP 23996 in Na+ buffer, which did not label this area) at stage 2 (pre-convulsive stage) and one week, but not one month, after stage 5 (generalized motor seizures). In contrast to somatostatin receptor binding, no alterations of the messenger RNA levels for sst1-sst5 receptors were found either at stage 2 or at stage 5. Similarly, no changes in receptor binding or messenger RNA levels were observed in the brain of rats which experienced a single afterdischarge. The present study shows a significant and selective decrease of somatostatin-1 receptor binding sites in the dentate gyrus of kindled rats. This is part of the plastic changes induced by kindling and may contribute to the increased sensitivity for the induction of generalized seizures during kindling.

Functional activation of somatostatin- and neuropeptide Y-containing neurons in the entorhinal cortex of chronically epileptic rats.

The in vitro release of somatostatin and neuropeptide Y, their tissue concentration and immunocytochemical pattern were examined in the entorhinal cortex of chronically epileptic rats. A systemic administration of 12 mg/kg kainic acid causing generalized tonic-clonic seizures for at least 3 h after injection was used to induce, 60 days later, a chronically enhanced susceptibility to seizures in the rats. The release of both peptides under depolarizing conditions was significantly reduced by 15% on average from slices of the entorhinal cortex two days after kainic acid-induced status epilepticus. At 60 days, the spontaneous and 30 mM KCl-induced release of somatostatin was significantly enhanced by 30% on average. The release induced by 100 mM KCl was raised by 70%. The spontaneous, 30 mM and 100 mM KCl-induced release of neuropeptide Y from the same slices was increased, respectively, by 120%, 76% and 36%. The late changes were associated with an increased tissue concentration of neuropeptide Y but not of somatostatin. This was confirmed by immunocytochemical evidence showing that neuropeptide Y-, but not somatostatin-immunoreactive neurons were increased in the entorhinal cortex of kainic acid-treated rats. These results indicate that neurotransmission mediated by somatostatin and neuropeptide Y, two peptides previously shown to play a role in limbic epileptogenesis, is enhanced in the entorhinal cortex of chronically epileptic rats.

Somatostatin release is enhanced in the hippocampus of partially and fully kindled rats.

The release of somatostatin (somatostatin-like immunoreactivity) from hippocampal slices during the development of hippocampal kindling in rats was measured under resting and depolarizing conditions. Preliminary experiments in naive rats showed that the spontaneous efflux of somatostatin (4.0 +/- 0.3 fmol/ml every 10 min) was independent of external Ca2+ but was reduced to 71.5 +/- 6% of baseline (P < 0.05) during 20 min incubation with 5 microM tetrodotoxin. Neuronal depolarization with 25, 50 and 100 mM KCl induced a Ca(2+)-dependent somatostatin release, respectively 4.3 +/- 0.4, 16.7 +/- 1.6 and 22.0 +/- 1.3 times baseline (P < 0.01). Veratridine caused a dose-dependent Ca2+ and tetrodotoxin (5 microM) sensitive release ranging from 6.5 +/- 0.1 to 13.0 +/- 1.4 times baseline at 1.4 microM and 50 microM respectively (P < 0.01). One week after the last of three consecutive stage 5 seizures (full seizure expression) or 48 h after the last stage 2 stimulation (preconvulsive stage), 50 mM KCl-induced somatostatin release was significantly higher (1.8 +/- 0.1, P < 0.01) than in shams (animals implanted with electrodes but not stimulated) in the stimulated and contralateral hippocampus. Somatostatin release measured under resting conditions was increased by 1.5 times in the stimulated hippocampus at stage 2 (P < 0.05) and by 2.2 and 1.7 times in both hippocampi at stage 5 (P < 0.01). Forty-eight hours after the induction of a single afterdischarge no significant changes were found in either spontaneous or 50 mM KCl-induced release of somatostatin.(ABSTRACT TRUNCATED AT 250 WORDS)