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Selective depletion of nerve growth factor receptor-bearing neurons in the basal forebrain cholinergic system nuclei by the immunotoxin 192 IgG-saporin offers a new and highly useful tool for the study of the role of the forebrain cholinergic system in cognitive functions. In the present study, we have tested the effects of 192 IpG-saporin in an operant delayed matching-to-position task which has previously been used to discriminate between delay-dependent learning impairments and delay-independent disturbances of non-mnemonic processes. Rats were first trained to criterion performance and then received intraventricular injections of 5 microg of 192 IgG-saporin 4 weeks prior to a second testing session. Rats with 192 IgG-saporin lesions displayed a significant delay-dependent decline in performance compared to normal controls, indicating a deficit in short-term memory. Administration of the muscarinic blocker scopolamine (0.5 mg/kg, i.p.) produced more pronounced impairment in the performance of the normal control rats across all delays, and induced further impairment also in animals with 192 IgG-saporin lesions. These effects were not observed following control injections of methyl scopolamine, suggesting that the impairment induced by scopolamine was due to the blockade of central muscarinic receptors. No improvement in performance was observed in either group following systemic treatment with the muscarinic cholinergic agonist arecoline (1.00 mg/kg). Biochemical and morphological analyses confirmed the selective and severe (>90-95%) depletion of cholinergic neurons throughout the septal-diagonal band area and the nucleus basalis region by the intraventricular 192 IgG-saporin treatment. Although the immunotoxin was observed to produce additional damage to the cerebellar Purkinje cells, no gross motor abnormalities were observed that could contribute to the effects on accuracy in the task used here. In conclusion, the results show that selective combined lesions of the basal forebrain cholinergic neurons in the septal-diagonal band area and nucleus basalis produce long-lasting impairments in short-term memory, thus providing further support for a role of this system in cognitive functions.

The immunotoxin 192 IgG-saporin induces an efficient and specific lesion of low-affinity nerve growth factor receptor-bearing cholinergic neurons in the basal forebrain. Intraventricular injection of 192 IgG-saporin, which caused a complete loss of cholinergic afferents to the hippocampus and neocortex and a partial denervation of amygdala and piriform cortex, was found to markedly facilitate the initial stages of seizure development in hippocampal kindling. In contrast, the progression of kindling process from focal to generalized seizures was not affected. In situ hybridization demonstrated that basal levels of brain-derived neutrotrophic factor messenger RNA in the hippocampal formation and piriform cortex were significantly decreased by the lesion, which also attenuated the seizure-induced increase of brain-derived neurotrophic factor messenger RNA expression in the hippocampus and frontal cortex. In the dentate gyrus, the 192 IgG-saporin lesion selectively reduced the upregulation of messenger RNAs for brain-derived neurotrophic factor exons I and III after a generalized seizure, whereas the increase of exon II messenger RNA was unchanged. The lesion abolished the seizure-evoked increase of nerve growth factor and TrkC messenger RNA levels and decrease of neutrophin-3 messenger RNA expression in dentate granule cells, while TrkB messenger RNA levels were not affected. We conclude that the basal forebrain cholinergic system (1) suppresses kindling epileptogenesis in the hippocampus, and (2) enhances both basal and seizure-evoked brain-derived neurotrophic factor synthesis in the hippocampal formation and some cortical areas through a specific pattern of activation of promoters within the brain-derived neurotrophic factor gene.

The distribution and the chemical identity of retrogradely single and double labeled neurons in the caudal raphe nuclei were analyzed in the rat following injection of two fluorescent tracers into the medial preoptic area and the ventral/intermediate grey of the lumbar spinal cord, and serotonin immunocytochemistry. The results suggest that (1) neurons in the caudal raphe nuclei exhibit highly collateralized axons, able to simultaneously innervate rostrally- and caudally-located targets; (2) a large proportion (40-50%) of the raphe-spinal projection does not contain serotonin, which by contrast is present in more than 70% of the neurons projecting to the medial preoptic area; (3) only a small fraction of the observed collateralized projection is serotonergic. Thus, multiple transmitter systems are likely to be involved in the diffuse ascending and descending influence arising from these nuclei.

Prolonged exposure of cultured cortical cells or cultured cerebellar granule cells to the residue 25-35 fragment of beta-amyloid peptide (beta AP), beta AP(25-35), induced neuronal apoptosis, as revealed by morphological analysis, fluorescent chromatin staining, and immunodetection of oligonucleosomes released from the nucleus into the cytoplasm. beta AP(25-35)-induced apoptosis was insensitive to ionotropic glutamate receptor antagonists but was substantially attenuated by the metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid. The neuroprotective action of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid was antagonized by (RS)-alpha-methyl-4-carboxyphenylglycine and was mimicked by (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (a selective agonist of mGluR2 and -3 subtypes) and by L-2-amino-4-phosphobutanoate and L-serine-O-phosphate (selective agonists of mGluR4, -6, and -7 subtypes). However, whereas all of these drugs behaved as neuroprotectants in cultured cortical cells, only L-2-amino-4-phosphobutanoate and L-serine-O-phosphate [and not (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine] reduced beta AP(25-35)-induced apoptosis in cultured cerebellar granule cells. The neuroprotective activity of mGluR agonists may be related to their ability to inhibit membrane Ca2+ conductance, because drugs that block voltage-sensitive Ca2+ channels, such as nimodipine or Co2+, could also attenuate beta AP(25-35)-induced apoptosis.

The elucidation of the functional role of the basal forebrain cholinergic system will require access to a highly specific and efficient cholinergic neurotoxin. Recently, selective depletion of the nerve growth factor (NGF) receptor-bearing cholinergic neurons in the rat basal forebrain and a dramatic loss of cholinergic innervation in the related cortical regions have been obtained following intraventricular injection of a newly introduced immunotoxin, 192 IgG-saporin. Here we extend these initial findings and report that administration of increasing doses (1.25, 2.5, 5.0 or 10 micrograms) of the 192 IgG-saporin conjugate into the lateral ventricles of adult rats induced dose-dependent impairments in the water maze task and passive avoidance retention, but only weak and inconsistent effects on locomotor activity. These behavioural changes were paralleled by a reduction in choline acetyltransferase activity in hippocampus and several cortical areas (up to 97%) and selective depletions of NGF receptor-positive cholinergic neurons in the septal-diagonal band area and nucleus basalis magnocellularis (up to 99%). By contrast, the non-cholinergic parvalbumin-containing neurons in the septum were completely spared, and other cholinergic projection systems (such as in the striatum, thalamus, brainstem and spinal cord) were unaffected even at the highest dose. The observed changes in the water maze and passive avoidance tasks, as well as the cholinergic cell loss, were maintained up to at least 8 months following the intraventricular injection of a single dose (5 micrograms) of the immunotoxin. The results confirm the usefulness of the 192 IgG-saporin toxin for selective and profound lesions of the basal forebrain cholinergic neurons and provide further support for a role of the basal forebrain cholinergic system in cognitive functions.

Gene expression in mammalian cells can be suppressed by oligonucleotides complementary to the target mRNA. This strategy was explored as a means of arresting translation of the prohormone precursor proopiomelanocortin (POMC), used as a model system of peptide messengers that are synthesized and released from endocrine and neuronal cells. The synthesis of the POMC-derived peptides adrenocorticotropin (ACTH) and beta-endorphin (beta-END) was markedly reduced by an oligodeoxynucleotide (ODN) complementary to a region of beta-END mRNA in AtT-20 cells, which retain many of the differentiated phenotypes of corticotrophs; this treatment did not affect the steady-state levels of POMC mRNA. Antisense ODN was stable in cell culture medium for 24 h, and cellular uptake was low (approximately 2.5% of the added ODN); however, the intracellular levels of the ODN were sufficient to form a ribonuclease-resistant duplex with complementary cellular mRNA. Addition of ODN to the cell culture did not affect the cellular levels of chromogranin A-(264-314)/pancreastatin or cell viability and proliferation, as evidenced by bromodeoxyuridine incorporation and ornithine decarboxylase activity. Microinfusion of the antisense ODN in the rat hypothalamic arcuate nucleus, where the majority of POMC-positive brain perikarya are located, significantly reduced ACTH- and beta-END-immunopositive neurons, and antisense ODN-treated rats showed substantially less of the grooming behavior usually observed in a novel environment.

The effect of amphetamine on striatal acetylcholine (ACh) release was studied by an in vivo intrastriatal microdialysis technique. Although we expected systemic amphetamine to inhibit baseline striatal ACh release, the opposite was found. In addition, we found that the amphetamine-induced striatal ACh release did not depend on nigrostriatal DA since 6-hydroxydopamine (6-OHDA) lesions had no effect on amphetamine-induced ACh release. Local intrastriatal injection of amphetamine via the microdialysis probe had no effect on striatal ACh release even when the probe was located more laterally in striatum to take advantage of the medial to lateral gradient of striatal ACh and D2 receptors. The hypothesis that amphetamine increased extracellular striatal ACh by increasing the release of biogenic amines besides dopamine was tested by pharmacological manipulations designed to specifically increase local striatal norepinephrine or serotonin levels. The serotonergic and noradrenergic manipulations had no effect on striatal ACh levels. These results indicate that amphetamine-induced release of ACh in striatum is mediated via distal brain regions that are functionally connected with the striatum.

The regulation of acetylcholine (ACh) release from cholinergic neurons transplanted to the hippocampus or neocortex was studied by microdialysis in awake rats. Fetal basal forebrain tissue was implanted as a cell suspension or solid graft into the fimbria-fornix-lesioned hippocampus, or as a cell suspension into the frontal cortex after excitotoxic lesion of the nucleus basalis. Several months after transplantation, microdialysis probes were implanted in areas of the hippocampus or frontal cortex reinnervated by the grafts. The grafts restored lesion-induced deficits in steady-state ACh release up to normal or above normal levels in both hippocampus and frontal cortex. The responses to KCl and tetrodotoxin suggested that the ACh release exhibited normal firing-dependent properties. By applying various behaviorally arousing stimuli that normally activate the basal forebrain projection systems, we wished to investigate the functional integration of the grafts in the host brain. In the hippocampus, sensory stimulation, immobilization stress and motor activity all resulted in increased release of graft-derived ACh amounting to 25-65% of the normal response. Variations in ACh levels during the day-night cycle was, however, not observed in the grafted rats. In the frontal cortex, immobilization enhanced the graft-derived ACh release (60% of normal response), whereas the response to sensory stimulation did not reach significance. Since the activity of the normal basal forebrain projection systems is under influence of monoaminergic brainstem afferents, we investigated the effects of systemic administration of amphetamine or apomorphine on ACh release in the hippocampus. Both drugs produced increases in graft-derived ACh release although the response was variable and less pronounced than normal. In conclusion, the graft-derived ACh release was affected by behavioral manipulations and catecholaminergic drugs that normally modify cholinergic septo-hippocampal and basalo-cortical activity. This strongly suggests a high degree of functional integration of the graft in the host brain allowing for a regulated release of transmitter that can be adjusted during ongoing behavior.

Levels of messenger RNA for nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and the tyrosine kinase receptors trkA, trkB and trkC have been studied using in situ hybridization in the rat brain 2 h and four weeks after kindling-induced seizures. Epileptiform activity evoked by hippocampal stimulation and exceeding 70 s lead to a concomitant and transient increase of brain- derived neurotrophic factor, nerve growth factor, trkB and trkC messenger RNA expression in dentate granule cells after both focal and generalized seizures. Brain-derived neurotrophic factor messenger RNA levels were also increased bilaterally in the CA1-CA3 regions, amygdala and the piriform, entorhinal, perirhinal, retrosplenial and temporal cortices after generalized seizures. The magnitude of the increases was similar throughout the development of kindling and in the fully kindled brain. No changes of trkA messenger RNA were observed. In amygdalar kindling, elevated brain-derived neurotrophic factor messenger RNA levels developed more rapidly in the amygdala-piriform cortex than after stimulation in the hippocampus but changes in the hippocampal formation were only seen in few animals. Intraventricular 6-hydroxydopamine or a bilateral fimbria-fornix lesion did not alter basal expression or seizure-evoked changes in messenger RNA levels for neurotrophins or trk receptors but increased the number of animals exhibiting elevated levels after the first stimulation, probably due to a prolongation of seizure activity. Both in sham-operated and fimbria-fornix-lesioned rats seizure activity caused a marked reduction of neurotrophin-3 messenger RNA levels in dentate granule cells. The results indicate that activation of the brain-derived neurotrophic factor gene, at least in dentate granule cells, is an "all-or-none" type of response and dependent on the duration but not the severity of seizures or the stage of kindling epileptogenesis. Changes in brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3 and trkB and trkC were observed concomitantly in the dentate gyrus, which suggests that seizure activity sets in motion a cascade of genomic events possibly mediated via a common mechanism. Since altered messenger RNA levels outside hippocampus were detected only for brain-derived neurotrophic factor, neurotrophin and trk gene expression in these regions seems to be regulated differently.

A monoclonal antibody to the low-affinity NGF receptor, 192 IgG, coupled to a cytotoxin, saporin, was recently introduced as an efficient selective neurotoxin for the NGFr-bearing cholinergic neurones in the rat basal forebrain. In the present study we report that an intracerebroventricular injection of this 192 IgG-saporin conjugate induces a severe, long-lasting spatial learning impairment, as assessed in the Morris water-maze task. This behavioural impairment was associated with 65-90% depletion of choline acetyltransferase activity (ChAT) in the hippocampus and cortex. ChAT activity associated with other cholinergic neurone systems in the brain (striatum, mesencephalon, spinal cord), was left virtually unaffected. This new immunotoxin holds great promise as a tool for selective and efficient lesions of the forebrain cholinergic system in functional and behavioural studies.

The role of monoamines in the functional regulation of the septo-hippocampal cholinergic system was studied using in vivo microdialysis of acetylcholine (ACh) release in the hippocampus of awake unrestrained rats. Systemic administration of the dopamine receptor agonist apomorphine (2.0 mg/kg) resulted in a 170% increase in hippocampal ACh overflow. Similarly the catecholamine-releasing agent amphetamine (2.5 mg/kg) produced a 400% increase in ACh overflow. The effect induced by amphetamine, but not that of apomorphine, was blocked in animals pretreated with the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine (AMPT). The effect of amphetamine on ACh release was reduced by 75% after a 6-hydroxydopamine (6-OHDA) lesion of the ventral tegmental area (VTA) but was not affected by 6-OHDA lesions of the noradrenergic dorsal and ventral bundles. However, baseline ACh overflow was increased by 130% by the dorsal and ventral bundle lesions. The serotonin-releasing agent p-chloroamphetamine (2.5 mg/kg) produced a 160% increase in hippocampal ACh release, and this effect was enhanced after a 5,7-dihydroxytryptamine (5,7-DHT) lesion of the serotonin projection system. The results show that surgical or pharmacological manipulations of the ascending brainstem monoaminergic systems, which innervate wide areas of the forebrain, including the septum and the hippocampal formation, have pronounced effects on septo-hippocampal cholinergic activity. Thus, the present data provide support for the view that information regarding behavioral state and arousal is conveyed to the septo-hippocampal system via ascending monoaminergic systems.

In this study, we utilized a double retrograde axonal tracing technique to investigate the possible existence of collateralized axonal projections from raphe nuclei 'pontis' and 'magnus' to both medial preoptic area (MPA) and cervical spinal cord (C1-C2). Following microinjections of fluorescent tracers Fast blue (FB) and Diamidino yellow (DY) within MPA and C1-C2, substantial numbers of FB and DY single-labeled neurons, as well as FB-DY double-labeled neurons have been found within raphe nuclei 'pontis' and 'magnus'.

In this study we utilized a double retrograde axonal tracing technique to investigate the possible existence of collateralized axonal projections of subcoeruleus area neurons to both raphe pontis nucleus and hypothalamic zona incerta. Following microinjections of fluorescent tracers (Fast blue (FB) and Diamidino yellow (DY] within raphe pontis and zone incerta, substantial numbers of double-labeled branched neurons have been found within the subcoeruleus area.

In this study we utilized a double retrograde axonal tracing technique to investigate the possible existence of collateralized axonal projections of subcoeruleus area neurons to both 'medial preoptic area (MPA) and cervical (C1-C3) spinal cord'. Following microinjections of fluorescent tracers (Fast blue (FB) and Diamidino yellow (DY) within MPA and C1-C3, substantial numbers of FB and DY single-labeled neurons as well as FB-DY double-labeled branched neurons have been found within subcoeruleus area.

Afferent projections into rat supraoptic nucleus (SO) and paraventricular nucleus (PVH) were studied with two fluorescent retrograde neuronal tracers: Nuclear Yellow (NY) and Fast Blue (FB). FB labeled cells were found into paraolfactory area (ACB), basolateral amygdaloid nucleus (ABL), lateral hypothalamus (LH), ventromedial nucleus (VMH) and dorsomedial hypothalamic)nucleus (DMH), tegmental nucleus of the pons (TPO). NY labeled cells were found into posterior hypothalamus (PH), dorsal raphe nucleus (DR) and tegmental nucleus of the pons (TPO). Few double-labeled neurons were found into TPO. The finding of double labeled neurons in the TPO is of particular interest. This latter nucleus is classically connected to the cerebellum trough the olivary nucleus and can serve as liaison for transmission of messages, e.g. vestibular, regarding spacial position variations. We can conclude affirming that quite a lot of informations of diverse nature converges on the SO and PVH nuclei that by efferent pathways may regulate hypothalamic neurosecretion, thus modifying the animal's behaviour response to different external stimuli.

In this study, we have examined the role of the dorsomedial (DMH), ventromedial (VMH) and arcuate (ARH) nuclei of the hypothalamus in the control of hypothalamic and pituitary immunoreactive (ir) dynorphin (Dyn) A and ir-Dyn B in the rat, by evaluating the effect of discrete, bilateral radiofrequency lesions in these structures. Lesions limited to the VMH reduced the content of ir-Dyn in the anterior pituitary but not in the neurointermediate lobe or in the hypothalamus. Gel chromatographic analysis of anterior pituitary extracts confirmed that ir-Dyn is mainly associated with high molecular weight forms containing Dyn A and Dyn B in their sequence. Anterior pituitary extracts of VMH-lesioned rats displayed a clearly lower proportion of these forms. Destruction of the DMH affected only the hypothalamic content of ir-Dyn; ablation of the ARH did not cause any significant change. Our results suggest that ablation of the VMH may disrupt critical neuronal connections to the median eminence originating in this nucleus or crossing it and participating in control of the adenohypophyseal pool of ir-Dyn.