Septohippocampal Cholinergic Innervation Research Articles

Effects of p75NTR deficiency on cholinergic innervation of the amygdala and anxiety-like behavior.

The p75 neurotrophin receptor (p75NTR) is a low-affinity receptor that is capable of binding neurotrophins. Two different p75NTR knockout mouse lines are available either with a deletion in Exon III (p75NTRExIII-/- ) or in Exon IV (p75NTRExIV-/- ). In p75NTRExIII knockout mice, only the full-length p75NTR is deleted, whereas in p75NTRExIV knockout mice, the full-length as well as the truncated isoform of the receptor is deleted. Deletion of p75NTR has been shown to affect, among others, the septohippocampal cholinergic innervation pattern and neuronal plasticity within the hippocampus. We hypothesize that deletion of p75NTR also alters the morphology and physiology of a further key structure of the limbic system, the amygdala. Our results indicate that deletion of p75NTR also increases cholinergic innervation in the basolateral amygdala in adult as well as aged p75NTRExIII-/- and p75NTRExIV-/- mice. The p75NTRExIV-/- mice did not display altered long-term potentiation (LTP) in the basolateral amygdala as compared to age-matched control littermates. However, p75NTRExIII-/- mice display stronger LTP in the basolateral amygdala compared to age-matched controls. Bath-application of K252a (a trk antagonist) did not inhibit the induction of LTP in the basolateral amygdala, but reduced the level of LTP in p75NTRExIII-/- mice to levels seen in respective controls. Moreover, p75NTRExIII-/- mice display altered behavior in the dark/light box. Thus, deletion of p75NTR in mice leads to physiological and morphological changes in the amygdala and altered behavior that is linked to the limbic system.

Mechanism‐Based Approaches for the Reversal of Drug Neurobehavioral Teratogenicity

Understanding the mechanism of neurobehavioral teratogenicity is the primary prerequisite for reversal of the defect. Progress in such studies can be best achieved if the investigation focuses on behaviors related to a specific brain region and innervation. Our model focused on teratogen-induced deficits in hippocampus-related eight-arm and Morris maze behaviors. Different "cholinergic" teratogens, mainly heroin, induced both pre- and postsynaptic hyperactivity in the hippocampal cholinergic innervation that terminated in desensitization of Protein Kinase C (PKC) isoforms to cholinergic receptor stimulation. Understanding this mechanism enabled its reversal with a pharmacological therapy-nicotine infusion. Studies by others provided similar findings by targeting the deficits respective to the model investigated. Consistently, destruction of the A10-septal dopaminergic pathways that downregulate the septohippocampal cholinergic innervation ameliorated maze performance. Grafting of embryonic differentiated cholinergic cells or neural progenitors similarly reversed the biochemical/molecular alterations and the resulting deficits. Reversal therapies offer a model for the understanding of neurobehavioral teratogenicity and, clinically, offer a model for potential treatment of these deficits. Whereas neural progenitor grafting appears to be the most effective treatment, pharmacological reversal with nicotine infusion seems to possess the most feasible and immediate therapy for neurobehavioral birth defects produced by various teratogens, including drugs. This is true even though the effect of pharmacological therapies is diffuse, affecting multiple areas of the brain. "Everybody is talking about the weather but nobody does anything about it." (Mark Twain).

Disruption of the development of cholinergic-induced translocation/activation of PKC isoforms after prenatal heroin exposure

Prenatal exposure of mice to heroin resulted in behavioral deficits present at adulthood, and related to septohippocampal cholinergic innervation accompanied by both pre- and postsynaptic cholinergic hyperactivity; including an increase in membrane PKC activity, and a desensitization of PKC to cholinergic input, which correlated highly with the behavioral performance, and was reversed by cholinergic grafting. The effect was shown in the behaviorally relevant PKCγ and β whereas the less behaviorally relevant PKCα isoform was not affected. The present study was designed to establish the effect of heroin exposure on the expression of the PKC isoforms level and on the more functionally relevant cholinergic translocation/activation of the isoforms throughout postnatal development. The hippocampi of mice pups, exposed to heroin transplacentally, were assayed after incubation with carbachol for PKC isoforms on postnatal days (PN) 1, 7, 14, 21, 30 and 50. Prenatal heroin exposure increased basal PKCγ, β and α levels. PKCγ and α levels returned to control levels on PN50. While in PKCβ, this increase lasted until PN50. Translocation/activation of the PKC isoforms γ and β by cholinergic receptor stimulation was present from PN1, concurrent with the presence of the isoforms. Prenatal exposure to heroin completely abolished the translocation/activation throughout the entire postnatal development. This defect was shown from the very beginning, PN1, the day when the PKC isoforms appear. The results suggest that the PKCγ and β isoforms are functional concurrent with their developmental appearance. Unlike findings on some other teratogens, the prenatal heroin effect on the isoforms function is similar throughout postnatal development.

Prenatal heroin exposure alters cholinergic receptor stimulated activation of the PKC$beta;II and PKC$gamma; isoforms

Prenatal exposure of mice to heroin (SC injection of 10 mg/kg to the dams on gestational days 9–18) resulted at adulthood in behavioral deficits related to septohippocampal cholinergic innervation accompanied with both presynaptic and postsynaptic cholinergic hyperactivity; including an increase membrane PKC activity, and a desensitization of PKC to cholinergic input which were highly correlated with the behavioral performance and were reversed by cholinergic grafting. Therefore, we studied the receptor induced activation of the behaviorally relevant PKCγ and PKCβII isoforms and the less behaviorally relevant PKCα isoform. Time course studies revealed peak translocation after 40 min incubation with carbachol for PKCγ (110% increase from basal, i.e. no carbachol level, P < 0.01), 30 min for phosphorylated PKCβII (130%, P < 0.05) and 5 min for non-phosphorylated PKCβII (64%, P < 0.05) with no peak for alpha. Prenatal heroin abolished the translocation of PKCγ and PKCβII while PKCα remained unaffected. A decrease occurred in basal phosphorylated membrane (−45%, P < 0.01) and cytosol-associated (−29%, P < 0.01) PKCβII, in membrane-associated non-phosphorylated PKCβII (−32%, P < 0.01) and PKCγ (−25%, P < 0.01) and in cytosolic PKCα (−27%, P < 0.01), while membrane-associated PKCα was slightly increased (11%, P < 0.05). The results suggest that prenatal heroin disrupts cholinergic receptor induced PKC translocation and activation with the underlying mechanism of neuroteratogenicity potentially lying in the PKCγ and PKCβII, while PKCα remains unaffected.

Prenatal heroin exposure alters cholinergic receptor stimulated activation of the PKCβII and PKCγ isoforms

Prenatal exposure of mice to heroin (SC injection of 10 mg/kg to the dams on gestational days 9–18) resulted at adulthood in behavioral deficits related to septohippocampal cholinergic innervation accompanied with both presynaptic and postsynaptic cholinergic hyperactivity; including an increase membrane PKC activity, and a desensitization of PKC to cholinergic input which were highly correlated with the behavioral performance and were reversed by cholinergic grafting. Therefore, we studied the receptor induced activation of the behaviorally relevant PKCγ and PKCβII isoforms and the less behaviorally relevant PKCα isoform. Time course studies revealed peak translocation after 40 min incubation with carbachol for PKCγ (110% increase from basal, i.e. no carbachol level, P < 0.01), 30 min for phosphorylated PKCβII (130%, P < 0.05) and 5 min for non-phosphorylated PKCβII (64%, P < 0.05) with no peak for alpha. Prenatal heroin abolished the translocation of PKCγ and PKCβII while PKCα remained unaffected. A decrease occurred in basal phosphorylated membrane (−45%, P < 0.01) and cytosol-associated (−29%, P < 0.01) PKCβII, in membrane-associated non-phosphorylated PKCβII (−32%, P < 0.01) and PKCγ (−25%, P < 0.01) and in cytosolic PKCα (−27%, P < 0.01), while membrane-associated PKCα was slightly increased (11%, P < 0.05). The results suggest that prenatal heroin disrupts cholinergic receptor induced PKC translocation and activation with the underlying mechanism of neuroteratogenicity potentially lying in the PKCγ and PKCβII, while PKCα remains unaffected.

Functional changes after prenatal opiate exposure related to opiate receptors' regulated alterations in cholinergic innervation.

Opioid drugs act primarily on the opiate receptors; they also exert their effect on other innervations resulting in non-opioidergic behavioural deficits. Similarly, opioid neurobehavioural teratogenicity is attested in numerous behaviours and neural processes which hinder the research on the mechanisms involved. Therefore, in order to be able to ascertain the mechanism we have established an animal (mouse) model for the teratogenicity induced by opioid abuse, which focused on behaviours related to specific brain area and innervation. Diacetylmorphine (heroin) and not morphine was applied because heroin exerts a unique action, distinguished from that of morphine. Pregnant mice were exposed to heroin (10 mg/kg per day) and the offspring were tested for behavioural deficits and biochemical alterations related to the septohippocampal cholinergic innervation. Some studies employing the chick embryo were concomitantly added as a control for the confounding indirect variables. Prenatal exposure to heroin in mice induced global hyperactivation both pre- and post-synaptic along the septohippocampal cholinergic innervation, including basal protein kinase C (PKC) activity accompanied by a desensitization of PKC activity in response to cholinergic agonist. Functionally, the heroin-exposed offspring displayed deficits in hippocampus-related behaviours, suggesting deficits in the net output of the septohippocampal cholinergic innervation. Grafting of cholinergic cells to the impaired hippocampus reversed both pre- and post-synaptic hyperactivity, resensitized PKC activity, and restored the associated behaviours to normality. Consistently, correlation studies point to the relative importance of PKC to the behavioural deficits. The chick model, which dealt with imprinting related to a different brain region, confirmed that the effect of heroin is direct. Taken together with studies by others on the effect of prenatal exposure to opioids on the opioidergic innervation and with what is known on the opioid regulation of the cholinergic innervation, it appears that heroin exerts its neuroteratogenicity by inducing alterations in the opioidergic innervation, which by means of its regulatory action, attenuates the functional output of the cholinergic innervation. In our model, there was hyperactivity mostly of the post-synaptic components of the cholinergic innervation. However, the net cholinergic output is decreased because PKC is desensitized to the effect of the cholinergic agonist, and this is further evidenced by the extensive deficits in the related behaviours.

Hilar neuropeptide Y interneuron loss in the aged rat hippocampal formation

Neuropeptide Y-immunoreactive (NPY-I) interneurons in the dentate gyrus are vulnerable to various insults, including septohippocampal cholinergic deafferentation. The present study examined whether a loss of NPY-I neurons occurs during aging, when the functional integrity of the septohippocampal pathway is thought to be compromised. Sets of male Long Evans rats (consisting of young and aged rats, with and without spatial learning impairments assessed by the Morris water maze) were examined. Light microscopic analysis revealed that hilar NPY-I neuronal number in matched dorsal sections was significantly decreased in aged compared to young rats. Ultrastructural analysis disclosed that the microenvironment (the types of processes apposed to the plasmalemmal surface) of NPY-I neurons also differed significantly between young and aged rats. In particular, a subgroup of NPY-I neurons, distinguished by a higher percentage of unmyelinated axon coverage of the plasmalemmal surface, was present in young, but not aged, rats. Neither the number nor the microenvironment of NPY-I neurons significantly differed between aged animals that were impaired versus unimpaired in spatial learning performance. To our knowledge these findings represent the first report of an age-associated decline in the number of a specific, neurochemically identified neuronal subpopulation within the hippocampal formation. Additionally, they closely parallel observations in 192 IgG-saporin-lesioned animals, suggesting that a distinct subgroup of NPY-I interneurons is particularly dependent on the viability of septohippocampal cholinergic innervation for its survival. Since neuronal loss was not correlated with performance, this alteration by itself does not appear to be sufficient to produce learning impairment.

Alterations in PKCγ in the mouse hippocampus after prenatal exposure to heroin: a link from cell signaling to behavioral outcome

Administration of heroin to pregnant mice evokes neurochemical and behavioral deficits consequent to disruption of septohippocampal cholinergic innervation, notably involving desensitization of the ability of cholinergic receptors to activate PKC activity. The present study further evaluates whether desensitization occurs specifically for the PKCγ isoform, the behaviorally relevant subtype, as compared to PKCα. Mice were exposed transplacentally to heroin on gestational days (GD) 9–18 via s.c. maternal injections (10 mg/kg per day). In young adulthood (50 days old), control offspring showed an increase in hippocampal cell membrane PKCγ after incubation with the muscarinic cholinergic receptor agonist, carbachol, indicative of translocation from the cytosol. Prenatal exposure to heroin eliminated this response, whereas basal PKCγ levels were unchanged. In contrast, PKCα, which is not related to heroin-induced behavioral deficits, did not show a loss of response. The present findings strongly point to abnormalities in the responsiveness of PKCγ as a mechanism underlying the neurobehavioral teratogenicity of heroin.

Neural grafting reverses prenatal drug-induced alterations in hippocampal PKC and related behavioral deficits

Administration of heroin or phenobarbital to pregnant mice evokes neurochemical and behavioral deficits consequent to disruption of septohippocampal cholinergic innervation. The present study evaluates the relationship between the drug-induced biochemical changes and the behavioral deficits, applying two different approaches: neural grafting and within-individual correlations of biochemistry and behavior. Mice were exposed transplacentally to phenobarbital or heroin on gestational days 9–18 and tested in adulthood. Drug-exposed mice displayed impaired radial arm maze performance, increases in presynaptic choline transporter sites (monitored with [ 3H]hemicholinium-3 binding), upregulation of membrane-associated protein kinase C (PKC) activity, and desensitization of the PKC response to a cholinergic agonist, carbachol. Grafting of cholinergic cells to the impaired hippocampus reversed the behavioral deficits nearly completely and restored basal PKC activity and the PKC response to carbachol to normal; the drug effects on hemicholinium-3 binding were also slightly obtunded by neural grafting, but nevertheless remained significantly elevated. There were significant correlations between the performance in the eight-arm maze and both basal PKC activity and PKC desensitization, and to a lesser extent, between behavioral performance and hemicholinium-3 binding. Taken together, these findings indicate an inextricable link between the biochemical effects of prenatal drug exposure on the PKC signaling cascade and adverse behavioral outcomes. The ability of neural grafting to reverse both the drug-induced changes in PKC and behaviors linked to septohippocampal cholinergic function suggest a mechanistic link between this signaling pathway and neurobehavioral teratology caused by heroin or phenobarbital.

Ventral hippocampal ibotenic acid lesions block chronic nicotine-induced spatial working memory improvement in rats

Chronic nicotine infusions have been found to significantly improve working memory performance in the radial-arm maze. This effect is blocked by co-infusions of the nicotinic antagonist mecamylamine. Acute nicotine injections also improve working memory performance in the radial-arm maze. This effect is also blocked by mecamylamine co-administration. Recent local infusions studies have demonstrated the importance of the ventral hippocampus for nicotinic involvement in memory. Local infusions of mecamylamine, DHβE or MLA impair working memory performance on the radial-arm maze. The current study was conducted to determine the importance of the ventral hippocampus for the chronic effects of nicotine. Rats were trained on the working memory task in an eight-arm radial maze. After acquisition they underwent either infusions of ibotenic acid lesions or vehicle infusions and received subcutaneous implants of osmotic minipumps that delivered either nicotine at a dose of 5 mg kg −1 day −1 or vehicle in a 2×2 design. The rats then were given 2 days of recovery and were tested on the radial-arm maze three times per week for the next 4 weeks. As seen in previous studies, in the sham lesioned group nicotine infusions caused a significant improvement in choice accuracy. In contrast no nicotine-induced improvement was seen in the rats after ibotenic acid lesions of the ventral hippocampus. The effect of nicotine was blocked even though this lesion did not cause a deficit in performance. Previous work showed that chronic nicotine infusion still caused a significant improvement in working memory performance in the radial-arm maze after knife-cut lesions of the fimbria–fornix carrying the septo-hippocampal cholinergic innervation. Thus it appears that it is the postsynaptic nicotinic receptors in the ventral hippocampus which are critically important for the expression of the chronic nicotine induced working memory improvement.

Pre- and postsynaptic alterations in the septohippocampal cholinergic innervations after prenatal exposure to drugs

The present study was designed to evaluate possible presynaptic and postsynaptic alterations in the hippocampal cholinergic innervations that account for the hippocampus-related behavioral deficits found after prenatal drug exposure. Mice were prenatally exposed to either phenobarbital or heroin. On postnatal day 50, the hippocampi were removed and protein kinase C (PkC) activity, the amounts of Gi, Go, and Gq guanosine 5’-triphosphate binding proteins (G-proteins), and choline transports were determined. Basal PkC activity was higher than control levels in both phenobarbital and heroin treated mice, by 41% and 35%, respectively. The increase of PkC activity in response to carbachol was impaired in both treatment groups: in control mice, membrane PkC activity in hippocampal slices increased by 40%–50%, while no such response, or even slight reduction in PkC activity, was observed in the drug-exposed offspring. A significant increase was found in Gi and Gq G-proteins (18%–21%) in mice exposed to phenobarbital or to heroin compared with control levels. The amount of choline transporters, determined by hemicholinium binding, increased by 70% compared with the control level in mice prenatally exposed to heroin, and increased by 71% in mice prenatally exposed to phenobarbital. The alterations in basal and carbachol-stimulated hippocampal PkC activity after prenatal drug exposure may be related to an impairment in long-term potentiation (LTP); which plays an important role in hippocampal related behavioral abilities, changes in which are caused by prenatal drug exposure.

Glucocorticoid Facilitation of Cholinergic Development in the Rat Hippocampus

The role of endogenous glucocorticoids in facilitating the postnatal innervation of septohippocampal cholinergic projections was examined. Septohippocampal cholinergic innervation was determined using two methods. One method involved measuring the optical density of acetylcholinesterase, a marker of cholinergic fibres in the hippocampus. In the other method, acetylcholinesterase-positive fibre counts were made in the hippocampus. Both methods revealed that 14-day-old rats adrenalectomized at 10 days of age have significantly lower densities of acetylcholinesterase in the hippocampal dentate gyrus molecular layer and in the regio inferior when compared to sham-operated control rats. This reduction in hippocampal acetylcholinesterase did not occur when 10-day-old adrenalectomized rats were either injected daily with exogenous corticosterone (0.3 mg/100 g body weight) or when adrenalectomy was conducted at later postnatal ages. In addition, unlike the developing hippocampus, the basolateral nucleus of the amygdala, which is also highly innervated by cholinergic fibres, showed no significant changes in acetylcholinesterase density after adrenalectomy. These observations suggest that glucocorticoids play an important role in supporting the development of cholinergic projections to the hippocampus. Cholinergic innervation of the hippocampus appears especially sensitive to the action of glucocorticoids occurring before the conclusion of the second postnatal week. Furthermore, this glucocorticoid influence is directed rather specifically to the hippocampus in comparison to the basolateral amygdala.

Rat brain corticosteriod receptors are modulated by septo-hippocampal cholinergic innervation.

Brain corticosteroid receptors are regulated by neural control as well as by adrenal hormones. In this study we tested the effect of septo-hippocampal cholinergic lesions on the binding capacity of corticosteroid receptors. Rats were injected with ibotenic acid into the medial septum, and hippocampal type I and type II, and hypothalamic type II corticosteroid receptors were measured 15 days later. In lesioned animals the number of type I corticosteroid receptors in the hippocampus was increased and their affinity decreased. In the hypothalamus only an increase of type II corticosteroid receptors was observed. As expected, lesioned rats showed an increase in basal plasma corticosterone levels.

Behavioral deficits and related septohippocampal cholinergic alterations after prenatal exposure to heroin

Pregnant female mice were exposed to heroin on gestation days 9–18. On postnatal day 50, offspring showed marked deficits in the hippocampus related behaviors, eight-arm and Morris mazes. Alterations in the septohippocampal cholinergic innervations were studied as the possible mechanisms. We have found, presynaptically, that the binding of [ 3H]hemicholinium-3 (HC-3) (a reliable indicator of presynaptic cholinergic innervation) increased in the heroin exposed animals. The B max values of binding to muscarinic receptors (an indicator of postsynaptic cholinergic activity) increased as well. In agreement with this observation, carbachol induced formation of inositol phosphates (IP) similarly increased. Interestingly, we have also observed an increase in the levels of a subunit of several GTP binding proteins (Gai and Gaq). In addition, the basal level of Protein Kinase C (PKC) increased. However, PKC activity was unresponsive to acetylcholine agonist stimulation in the heroin treated animals, pointing to a possible explanation for the behavioral deficits.

Septohippocampal cholinergic changes after destruction of the A10-septal dopaminergic pathways

Mice were injected bilaterally into the septum with 6-hydroxydopamine and 6 weeks later the hippocampi were assayed for activity of choline acetyltransferase, muscarinic receptor binding capabilities and for formation of inositol phosphate in response to direct (carbachol) or presynaptically elicited (K +) stimulation of the postsynaptic receptors. Levels of dopamine in the septum were reduced by 70% in the lesioned animals and hippocampal choline acetyltranferase was elevated by the same amount. The B max of muscarinic binding was significantly reduced without changes in K d ; nevertheless, carbachol-induced stimulation of formation of inositol phosphate was unaffected. The response of K + was markedly elevated in the 6-hydroxydopamine-treated animals. Thus, the regulatory effect of A10-septal dopaminergic pathways on the septohippocampal cholinergic innervations is both on the presynaptic and postsynaptic levels.

Alterations in septohippocampal cholinergic innervations and related behaviors after early exposure to heroin and phencyclidine

Mice were exposed to diacetylmorphine (heroin) or phencyclidine (PCP) prenatally or neonatally. At a later age, they were tested for hippocampus-related behavioral deficits and concomitant alterations in the septohippocampal cholinergic innervations. Actually, this is an application of the previously established phenobarbital neuroteratogenicity model to heroin and PCP. Prenatal exposure was accomplished transplacentally by injecting the mother 10 mg/kg heroin or PCP on gestation days 9–18. Neonatal administrations were applied directly by injections of 10 mg/kg of either drug to the pups between neonatal days 2–21. At the age of 50 days, mice exposed to heroin and PCP prenatally exhibited a 107% and 159% increase in their muscarinic cholinergic receptors B max, respectively. Neonatal exposure to heroin or PCP caused an 83% and 76% increase in the receptors respectively. On the behavioral level, both prenatal and neonatal exposure to heroin or PCP reduced performance in the hippocampus related eight-arm maze and Morris mazes. Depending on the drug, the test and the period of drug administration, the reduction ranged between 10% and 75%. The results suggest that heroin and PCP induce alterations in the septohippocampal cholinergic innervations and in related behavioral performance. Further studies are necessary in order to connect the biochemical and behavioral events in causal relationships.

Dopaminergic denervation reverses behavioral deficits induced by prenatal exposure to phenobarbital

Mice were prenatally exposed to phenobarbital. As adults, these mice (B animals) were deficient in the hippocampally related eight-arm maze performance, a behavior apparently dependent on the integrity of the septohippocampal cholinergic pathways. Preliminary studies suggest possible parallel alterations in their hippocampal cholinergic innervations. The dopaminergic septal innervations are known to indirectly inhibit the septohippocampal cholinergic innervations. Consequently, the septal dopaminergic innervations of adult B mice were destroyed by 6-hydroxydopamine (6-OHDA). Mice treated with 6-OHDA had an improvement in maze performance which was most marked with increased experience. Concomitant increase in choline acetyltransferase (ChAT) was also demonstrated in these mice (79%, P < 0.001). Similar increase in ChAT could be demonstrated in control mice after 6-OHDA treatment, but the behavioral changes were small and did not reach statistical significance, possibly due to the ceiling effect of the studied behavior. Thus, the dopaminergic innervations in the septum regulate cholinergic activity and its related behaviors along the septohippocampal pathway, and thereby ameliorate behavioral deficits induced by early phenobarbital administration.