Postnatal development of muscarinic autoreceptors modulating acetylcholine release in the septohippocampal cholinergic system: II. Cell body region: septum

doi:10.1016/S0165-3806(98)00027-3

Developmental Brain Research

Volume 108, Issues 1–2, 15 June 1998, Pages 31-37

https://doi.org/10.1016/S0165-3806(98)00027-3 Get rights and content

Abstract

We studied the postnatal development of the release of acetylcholine (ACh) and of presynaptic, release-inhibiting muscarinic autoreceptors in the cell body region of the septohippocampal cholinergic pathway. To this end, septal slices (350 μm thick) from rats of various postnatal ages (postnatal day 3 [P3] to P16) were preincubated with [³H]choline and stimulated twice (S₁, S₂: 360 pulses, 2 ms, 3 Hz, 60 mA) during superfusion with physiological buffer containing hemicholinium-3 (10 μM). In parallel, the activities of hemicholinium-sensitive high-affinity choline uptake (HACU, in synaptosomes) and of choline acetyltransferase (ChAT, in crude homogenates) were determined as markers for the development of cholinergic functions. In septal slices preincubated with [³H]choline, the electrically evoked overflow of ³H at S₁ increased from 0.31% (P3) to 2.10% of tissue ³H (P16), the latter value being still lower than that of septal slices from adult rats (3.46% of tissue ³H). Already at P3, the evoked overflow of ³H was Ca²⁺-dependent and sensitive to tetrodotoxin, indicating an action potential-evoked exocytotic mechanism of ACh release early after birth. Presence of the muscarinic agonist oxotremorine (1 μM) significantly inhibited the evoked ACh release in septal slices beginning from P5: no significant effect was detectable at P3. The ACh esterase inhibitor physostigmine (1 μM) exhibited significant inhibitory effects from P13 onwards. The muscarinic antagonist atropine (1 μM) enhanced the evoked ACh release only in septal tissue from adult rats. The specific activities of HACU, or ChAT showed a 2- or 8-fold increase, respectively, from P3 to P16. In conclusion, presynaptic cholinergic functions seem to develop almost in parallel both in the cell body and the target area of the septohippocampal projection: also in the septal region nerve terminals on axon collaterals are endowed very early (at least at P3) with the apparatus for action potential-induced, exocytotic release of ACh. In contrast, the appearance of feedback inhibition via presynaptic muscarinic autoreceptors is delayed. Autoinhibition due to endogenously released ACh can be detected only later, most probably when endogenous ACh concentrations in the septal nuclei have reached a threshold value.

Introduction

The neuroanatomical organization of the septal region, as well as the pre- and postnatal development of fiber connections to and from the septum have been extensively studied in the past [see Ref. [12]]. These studies support the view of the septal area as an interface between limbic telencephalic areas, on the one hand, and hypothalamic and brain stem areas on the other: thereby, regions associated with cognition and motivation are linked to areas related to endocrine and autonomic functions [12].

One major and well-known efferent projection of the medial septal nuclei and the nuclei of the diagonal band of Broca (MSDB) is the septohippocampal cholinergic connection [see Ref. [12]]. By means of monoclonal antibodies against choline acetyltransferase (ChAT), not only cholinergic cell bodies but also cholinergic nerve terminals have been detected in the MSDB [2]. These ChAT positive cholinergic nerve endings may arise either from axon collaterals of septohippocampal and/or intrinsic septal cholinergic neurons [see Refs. 3, 12], or from cholinergic projection neurons to the septum, which, however, are still a matter of debate [see Ref. [9]]. It has been proposed that the ChAT-positive nerve endings in the medial septum form synapses mainly with non-cholinergic, probably GABAergic cells [2].

Although neuroanatomical studies firmly support the presence of cholinergic nerve endings in the septal region, there are only a few studies on the main function of these axon terminals, i.e., the release of its neurotransmitter ACh and its modulation via presynaptic receptors. For instance, experiments on septal slices have shown that stimulation with high K⁺ concentrations induced a release of ACh, which was Ca²⁺-dependent and increased by atropine, suggesting a presynaptic autoreceptor-mediated negative feedback control [14]. Moreover, N-methyl-d-aspartate- (NMDA) evoked release of ACh from septal slices was shown to be tetrodotoxin- (TTX) sensitive, possibly indicating release from axon terminals that depended on Na⁺-induced action potentials [16]. Finally, an in vivo microdialysis study also provided evidence for the occurrence of presynaptic muscarinic autoreceptors on nerve terminals in the medial septal area [15]. As described in more detail in the accompanying paper [7], at least four muscarinic receptor types have been characterized pharmacologically. In the rat brain, muscarinic autoreceptors, which are localized on cholinergic axon terminals and inhibit ACh release, seem to belong to the M₂ and/or the M₄ type [for references, see Ref. [8]].

In the preceding paper [7], we studied the postnatal development of acetylcholine (ACh) release and its modulation via presynaptic muscarinic autoreceptors [see Ref. [19]] in the hippocampus. The main observation was that the appearance of muscarinic autoreceptors in the hippocampal region lagged behind that of the apparatus for exocytotic ACh release [7]. Therefore, the aim of the present investigation was to follow the development of cholinergic presynaptic functions also in the cell body region of this projection, the septal area. Specifically, we wanted to answer the following questions: (1) Does the development of neurotransmitter-specific presynaptic functions of cholinergic neurons, like high-affinity choline uptake (HACU) or choline acetyltransferase (ChAT), follow a time course similar to that in the hippocampus? (2) Does the electrically-evoked release of ACh in septal tissue possess properties similar to those in the axon terminal region, and is the developmental time course comparable? (3) Finally and most importantly, is it possible to demonstrate an autoreceptor-mediated inhibition of ACh release in this region, and if so, when do they become detectable?

Section snippets

Electrically evoked [³H]ACh release

Adult female Wistar rats (250–300 g b.wt.) or Wistar rats of various postnatal ages (P3 to P16) were decapitated. The brain was rapidly removed and placed with its dorsal surface on a cooled (4°C) metal block, thus exposing the ventral surface. The septal region was dissected according to Shahar et al. [18], with minor modifications. In brief, two transverse cuts were made through the forebrain: one rostral to the optic chiasm and a second through the optic chiasm, at the level of the rostral

Results

As in the preceding study [7], the time course of postnatal changes in the density of the cholinergic innervation of the rat septal region was studied by using two biochemical parameters: (1) the hemicholinium-sensitive high-affinity uptake of [³H]choline (HACU) into a vesicular fraction (corresponding to a crude synaptosomal fraction, P₂) prepared from this tissue, and (2) the activity of choline acetyltransferase (ChAT) in crude homogenates of septal tissue. For comparison, in this (and all

Discussion

The present study was undertaken to investigate properties and postnatal development of electrically-evoked ACh release, as well as of its autoreceptor-mediated modulation, in septal slices of the rat. Moreover, our interest was directed at the comparison of the observations from the cell body region of the septohippocampal projection, the septum, with those of the axon terminal region, the hippocampus [7].

Comparison of the postnatal time courses of the presynaptic cholinergic markers HACU and

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 505).

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Furthermore, acetylcholine (ACh) is one of the brain neurotransmitters [9] involved in learning, memory and attention processes [10–12]. The septo-hippocampal cholinergic connection is one major and well-known efferent projection of the MS and the vertical limb of the diagonal band of Broca (MS/VDB) [13–18] which has an important role for learning and memory [19,20]. There is also a suggestion that cholinergic projections from MS to the hippocampus could modulate hippocampal memory process [21].
The anatomical connections of septum and hippocampus and the influence of cholinergic and glutamatergic projections in these sites have been reported. In the present study, the effect of pre-training intra-dorsal hippocampal (CA1) and intra-medial septal (MS) administration of scopolamine, a nonselective muscarinic acetylcholine antagonist, and NMDA receptor agents and their interactions, on acquisition of memory have been investigated.
The animals were bilaterally implanted with chronic cannulae in the CA1 regions and in the medial septum. Animals were trained in a step-through type inhibitory avoidance task, and tested 24 h after training to measure step-through latency as memory retrieval.
Intra-CA1 or intra-MS injections of scopolamine (0.5, 1 and 2 μg/rat) and D-AP7 (a competitive NMDA receptor antagonist; 0.025, 0.05 and 0.1 μg/rat) reduced, while NMDA (0.125 and 0.25 μg/rat) increased memory. Intra-MS injection of a subthreshold dose of NMDA reduced scopolamine induced amnesia in the MS. However, similar injection of NMDA into CA1 did not alter scopolamine response when injected into CA1. Moreover, intra-MS or -CA1 injection of a subthreshold dose of NMDA did not alter scopolamine response in the CA1 or MS respectively. On the other hand, co-administration subthreshold doses of D-AP7 and scopolamine into CA1 and/or MS induced amnesia.
The cholinergic system between septum and CA1 are modulating memory acquisition processes induced by glutamatergic system in the CA1 or septum and co-activation of these systems in these sites can influence learning and memory.
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Recent EU/US pediatric legislation and FDA/EMEA guidelines recognize the potential differences in safety profiles of drugs in adults versus young patients. Hence safety studies are recommended to investigate key functional domains of e.g. the developing CNS. Methods: Selected psychoactive stimulants (caffeine, d-amphetamine, scopolamine) and depressants (baclofen, diazepam, haloperidol, chlorpromazine, imipramine, morphine) were characterized upon single administration with regard to behavioural parameters, locomotor activity, body temperature, pro-/anti-convulsive activity (pentylenetetrazole, PTZ), and nocifensive responses (hotplate) in neonatal (2 weeks), juvenile (4 weeks) and adult rats (8–9 weeks). Results: In vehicle-treated rats, behavioural patterns matured with age, locomotor activity and handling-induced rise in body temperature were enhanced, whereas PTZ convulsion threshold dose and nocifensive response latency decreased. Single test compound treatment elicited behavioural effects characteristic for psychoactive drugs with stimulating and depressing properties regardless of age. However, incidence of certain behaviours, and magnitude of effects on locomotor activity and body temperature varied with age and became generally more pronounced in adult rats. Pro-/anti-convulsive effects and delayed nocifensive responses did not differ between juvenile and adult rats. Conclusion: CNS effects of selected psychoactive reference compounds were qualitatively similar, but quantitatively different in neonatal, juvenile and adult rats.
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As described in the Experimental Procedures section, the septal region of the rat brain was roughly subdivided into tissue pieces containing (i) the dorsal and intermediate LS nuclei, (ii) the MS nucleus and (iii) the horizontal and vertical limb of the DB. This dissection method has proven useful in previous experiments of our group (Disko et al., 1998, 1999; Gazyakan et al., 2000a,b; Birthelmer et al., 2003b). The posterior part of the septum containing the septo-fimbrial nucleus and the triangular septal nucleus was not included in this study.
5-HT released from serotonergic axon terminals in the septal nuclei modulates the activity of septal output neurons (e.g. septohippocampal cholinergic neurons) bearing somatodendritic 5-HT receptors. Therefore, we studied the mechanisms involved in the presynaptic modulation of 5-HT release in the lateral (LS) and medial septum (MS), and the diagonal band of Broca (DB). HPLC analysis showed that tissue concentrations of noradrenaline, dopamine and 5-HT were highest in DB (DB>MS>LS). Slices prepared from LS, MS and DB regions were preincubated with [³H]5-HT, superfused in the presence of 6-nitro-2-(1-piperazinyl)-quinoline (6-nitroquipazine) and electrically stimulated up to three times (first electrical stimulation period (S₁), S₂, S₃; 360 pulses, 3 Hz, 2 ms, 26–28 mA). In all septal regions the Ca²⁺-dependent and tetrodotoxin-sensitive electrically-evoked overflow of [³H] was inhibited by the 5-HT_1B agonist CP-93,129 and the α₂-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline tartrate (UK-14,304). Also the μ- and κ-opioid receptor agonists (d-Ala², N-Me-Phe⁴, glycinol⁵)-enkephalin (DAMGO) and [trans-(1S,2S(-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]-benzenacetamide hydro-chloride] (U-50,488H), respectively, acted inhibitory (although less potently), whereas the δ-opioid receptor agonist (d-Pen², d-Pen⁵)-enkephalin (DPDPE), the dopamine D₂ receptor agonist quinpirole and the adenosine A₁ receptor agonist N⁶-cyclopentyladenosine were all ineffective; the GABA_B receptor agonist baclofen had weak effects. All inhibitory effects of the agonists were antagonized by the corresponding antagonists (3-[3-(dimethylamino)propyl]-4-hydroxy-N-[4-(4-pyridinyl)phenyl]benzamide dihydrochloride (GR-55,562), idazoxan, naloxone, nor-binaltorphimine), which also significantly enhanced the evoked release of 5-HT at S₁. It is concluded that 5-HT release in septal nuclei of the rat is modulated by presynaptic 5-HT_1B autoreceptors, as well as by α₂-, μ- and κ-opioid heteroreceptors. All of these receptors seem to be under a tonic inhibitory influence of the corresponding endogenous agonists and show qualitatively comparable modulatory properties along the dorso-ventral distribution of the 5-HT terminals.
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It can be said that the different groups of neurons acquire the cholinergic property at different developmental stages according to the functions with which they are concerned. On the other hand, in the septohippocampal system, remarkable development of the cholinergic nature occur in a time course similar to that of the LDT/PPT cells (at the second postnatal week) (Bender et al., 1996; Disko et al., 1998). It has been reported that, in neonatal rats, eye opens at P15 (Jouvet-Mounier et al., 1970), orienting response to auditory stimulus develops rapidly at P14 (Kelly et al., 1987) and auditory brainstem response is first observed at P12 (Blatchley et al., 1987).
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Acetylcholine (ACh) release is modulated pre-synaptically by both muscarinic and nicotinic receptor-mediated processes. While muscarinic autoreceptors inhibit ACh release, nicotinic autoreceptors enhance ACh release and thus disruption of these processes could potentially affect cholinergic toxicity following exposure to anticholinesterases. Marked age-related differences in sensitivity to some organophosphorus (OP) anticholinesterases have been reported. We compared nicotinic autoreceptor function (NAF) during maturation and aging and evaluated its potential modulation by the common OP insecticide, chlorpyrifos (CPF). Cortical synaptosomes were pre-loaded with [³H]choline, superfused (0.6 ml/min) with physiological buffer and [³H]ACh release was evoked with potassium (KCl, 9 mM), with or without co-addition of exogenous ACh to stimulate nicotinic autoreceptors. Fractions of perfusate were subsequently collected and area under the curve (AUC) for [³H] was analyzed by scintillation counting. The difference in evoked release due to co-addition of exogenous ACh was defined as NAF. Under these conditions, atropine (ATR, 0.1 μM) appeared requisite for NAF; thus this muscarinic antagonist was subsequently added to all perfusion buffers. In synaptosomes from adult tissues, exogenous ACh (3–100 μM) significantly increased release in a concentration-dependent manner. The nicotinic antagonist mecamylamine (MEC, 100 μM) substantially reduced the potassium-evoked release elicited by co-addition of ACh (10 μM). Interestingly, the nicotinic agonists nicotine (NIC) and dimethylphenylpiperazinium (DMPP; 0.1–10 μM) had no effect on release. The active metabolite of CPF (i.e. chlorpyrifos oxon (CPO), 1–10 μM) inhibited NAF in vitro. Maturation-related expression of NAF was noted (AUC with co-addition of 10 μM ACh: 7-day rats, 7±6; 21-day rats, 44±6; 90-day rats, 196±37; 24-month rats, 173±52). NAF was substantially reduced (67–91%) 96 h after maximum tolerated dosages of CPF in adult and aged rats (279 mg/kg, sc) but not in juveniles (127 mg/kg, sc), even though AChE inhibition was similar among the age groups (>80%). Together these data suggest that NAF is differentially expressed during maturation and that this neuromodulatory process may be selectively altered by some OP insecticides, potentially contributing to age-related differences in response to AChE inhibitors. As NAF has been postulated to be activated under conditions of ‘impaired’ cholinergic function, selective alteration of this pre-synaptic process by OP anticholinesterases may be also important in age-related conditions associated with cholinergic hypofunction.
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In previous studies electrically-evoked release of acetylcholine in septal slices was demonstrated. The present experiment aimed at verifying if this release involved intrinsic neurons bearing p75^NTR receptors. Long-Evans rats sustained injections of 192 IgG-saporin into the medial septum/diagonal band of Broca (0.8 μg). Sham-operated rats served as controls. Two to 3.5 weeks later, the electrically-evoked release of acetylcholine ([³H]ACh) was measured in slices from the lateral septum (LS), medial septum (MS) and diagonal band of Broca (DBB). Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity, and monoamine concentrations were measured in the septum, cortex and hippocampus. The lesion extent was also assessed by ChAT immunostaining in a separate series of rats. In the septum, the number of ChAT-positive neurons was depleted dramatically (>90% at the level of the injection site). In the hippocampus, the lesions reduced ChAT and AChE activity by 91% and 84%, respectively. In the cortex, this reduction was weaker (−55% and −47%). In the septal region, the reduction was either weak or not significant. The evoked release of acetylcholine in septal slices was not reduced, except in the slices from the LS (−64%). The effects of physostigmine and atropine confirmed the presence of autoreceptors. Our data exclude that a major part of the acetylcholine released by MS and DBB slices derived from intrinsic neurons bearing p75^NTR receptors. In the LS, part of the released acetylcholine might be from projections of such neurons located in the LS, MS and/or DBB. These data also suggest that the MS and the DBB may be the target of extrinsic cholinergic innervation that does not bear p75^NTR receptors.

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Research reportPostnatal development of muscarinic autoreceptors modulating acetylcholine release in the septohippocampal cholinergic system: II. Cell body region: septum

Abstract

Introduction

Section snippets

Electrically evoked [3H]ACh release

Results

Discussion

Acknowledgements

Mol. Brain Res.

Dev. Brain Res.

Neurosci. Lett.

Brain Res.

Neurosci. Lett.

Eur. J. Pharmacol.

Neurosci. Lett.

Brain Res.

Neuroscience

Development of cholinergic and GABAergic neurons in the rat medial septum: different onset of choline acetyltransferase and glutamate decarboxylase mRNA expression

J. Comp. Neurol.

Research report
Postnatal development of muscarinic autoreceptors modulating acetylcholine release in the septohippocampal cholinergic system: II. Cell body region: septum

Electrically evoked [³H]ACh release