Evidence for the existence of non-GABAergic, cholinergic interneurons in the rodent hippocampus

doi:10.1016/S0306-4522(99)00525-4

Neuroscience

Volume 96, Issue 1, January 2000, Pages 27-31

https://doi.org/10.1016/S0306-4522(99)00525-4 Get rights and content

Abstract

Previous studies have revealed a small number of hippocampal interneurons immunoreactive for choline acetyltransferase, the acetylcholine-synthesizing enzyme. It remained an open question, however, whether these neurons represented a subgroup of inhibitory GABAergic neurons co-localizing acetylcholine. In this study, we have combined immunocytochemistry for choline acetyltransferase and in situ hybridization for glutamate decarboxylase messenger RNA, the GABA-synthesizing enzyme. None of the choline acetyltransferase-immunoreactive neurons in the various layers of the hippocampus proper and fascia dentata were found to co-localize glutamate decarboxylase messenger RNA. The lack of an in situ hybridization signal in these neurons is unlikely to result from the combination of the two labeling techniques. When combining in situ hybridization for glutamate decarboxylase messenger RNA with immunostaining for parvalbumin, a calcium-binding protein expressed by many GABAergic hippocampal interneurons, numerous double-labeled cells were observed.

These data provide neurochemical evidence for the existence of non-GABAergic, supposedly cholinergic non-principal cells in the hippocampus.

Section snippets

Tissue fixation

Four adult Sprague–Dawley rats from the stock of the Anatomical Institute, University of Freiburg, Germany, were used for the present study. Animals were deeply anesthetized with a mixture of 25% Ketavet (Parke-Davis, 100 mg/ml), 6% Rompun (Bayer) and 2.5% Vetranquil (Sanofi, 13.56 mg acepromacine/ml) at a dose of 2.5 ml/kg body weight, and were transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The brains were postfixed for 2 h in 4% paraformaldehyde, cryoprotected

A small number of choline acetyltransferase-immunoreactive neurons are present in the various hippocampal layers

Immunostaining for ChAT confirmed the presence of a low number of weakly immunoreactive non-principal neurons in the various layers of the hippocampus and dentate gyrus12., 14., 19. (Fig. 1, Fig. 2A–C). They had small ovoid or round cell bodies and gave rise to two or occasionally three primary dendrites. In a total of 45 randomly selected hippocampal sections from the four animals, altogether 243 ChAT-immunoreactive neurons were found (5.4 neurons per section on average). Of these cells, 142

Discussion

In the present study, we confirmed the existence of a low number of ChAT-immunopositive non-principal cells in the hippocampus and dentate gyrus.12., 14., 19. Moreover, we showed that these interneurons do not stain for GAD65 or GAD67 mRNA. In contrast, numerous PARV-immunoreactive hippocampal neurons, known to be GABAergic,4., 5. were double labeled for GAD mRNA. We conclude from these data that there are a few non-GABAergic, supposedly cholinergic interneurons in the hippocampus.

Acknowledgements

We thank Drs N. Tillakaratne and A. Tobin for providing us with the GAD probes. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 505).

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Differential expression of various subtypes of the cholinergic receptors, their expression in multiple neuronal types within a region, and the varying locations within a neuron (i.e., somatic, dendritic, synaptic etc.) orchestrate a manifold symphony of neuromodulation. A representative example of this intricacy and the differential functional geography in the brain can be found when examining and comparing the olfactory system (Bohnen et al., 2010; D'Souza and Vijayaraghavan, 2014; Hellier et al., 2010), visual system (Bouskila et al., 2016; Groleau et al., 2015; Yi et al., 2015), and hippocampus (Alger et al., 2014; Frotscher et al., 2000; McQuiston, 2014; Yi et al., 2015) in light of cholinergic signaling (Vijayaraghavan and Sharma, 2015). In the olfactory system, nAChR activation has the capability to screen signals of odor in such a way that weak inputs stand rejected while the strong signals pass through the abstract threshold, giving rise to the gain of function in the olfactory circuit (D'Souza and Vijayaraghavan, 2014; Spindle et al., 2018) such as odor discrimination (D'Souza and Vijayaraghavan, 2014; Hellier et al., 2010; Spindle et al., 2018), whereas in the visual cortex, differential functional expression of mAChRs has a role to play in neuronal synchrony and gamma oscillations to modulate the network output during perceptual learning (Groleau et al., 2015).
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Cotransmission of acetylcholine and GABA
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Like the cortex, the striatum has both local cholinergic interneurons and long-distance innervation from the brainstem cholinergic centers (Calabresi et al., 2000). The hippocampus and neighboring entorhinal cortex, however, are innervated by extrinsic cholinergic inputs from the medial septum and horizontal and diagonal limbs of the band of Broca (MSDB; Teles-Grilo Ruivo and Mellor, 2013) and by intrinsic cholinergic interneurons (Frotscher et al., 1986, 2000; Romo-Parra et al., 2003; Yi et al., 2015). In these systems, ACh can activate ionotropic, excitatory nicotinic acetylcholine receptors (nAChRs) or metabotropic muscarinic acetylcholine choline receptors (mAChRs).
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Acetylcholine (ACh) is well known to induce persistent γ-oscillations in the hippocampus when applied together with physostigmine, an inhibitor of the ACh degrading enzyme acetylcholinesterase (AChE). Here we report that physostigmine alone can also dose-dependently induce γ-oscillations in rat hippocampal slices. We hypothesized that this effect was due to the presence of choline in the extracellular space and that this choline is taken up into cholinergic fibers where it is converted to ACh by the enzyme choline-acetyltransferase (ChAT). Release of ACh from cholinergic fibers in turn may then induce γ-oscillations. We therefore tested the effects of the choline uptake inhibitor hemicholinium-3 (HC-3) on persistent γ-oscillations either induced by physostigmine alone or by co-application of ACh and physostigmine. We found that HC-3 itself did not induce γ-oscillations and also did not prevent physostigmine-induced γ-oscillation while washout of physostigmine and ACh-induced γ-oscillations was accelerated. It was recently reported that ChAT might also be present in the extracellular space (Vijayaraghavan et al., 2013). Here we show that the effect of physostigmine was prevented by the ChAT inhibitor (2-benzoylethyl)-trimethylammonium iodide (BETA) which could indicate extracellular synthesis of ACh. However, when we tested for effects of extracellularly applied acetyl-CoA, a substrate of ChAT for synthesis of ACh, physostigmine-induced γ-oscillations were attenuated. Together, these findings do not support the idea that ACh can be synthesized by an extracellularly located ChAT.

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Neuroscience

Abstract

Section snippets

Tissue fixation

A small number of choline acetyltransferase-immunoreactive neurons are present in the various hippocampal layers

Discussion

Acknowledgements

References (24)

Monoclonal antibodies directed against the calcium-binding protein parvalbumin

Cell Calcium

Two genes encode distinct glutamate decarboxylases

Neuron

Cholinergic neurons in the rat hippocampus do not compensate for the loss of septohippocampal cholinergic fibers

Neurosci. Lett.

Organization and morphological characteristics of cholinergic neurons: an immunocytochemical study with a monoclonal antibody to choline acetyltransferase

Brain Res.

Choline acetyltransferase-immunoreactive neurons intrinsic to rodent cortex and distinction from acetylcholinesterase-positive neurons

Neuroscience

An immunocytochemical study of choline acetyltransferase-containing neurons and axon terminals in normal and partially deafferented hippocampal formation

Brain Res.

Cholinergic neurons in the rat central nervous system demonstrated by in situ hybridization of choline acetyltransferase mRNA

Neuroscience

Cholinergic systems in mammalian brain and spinal cord

Prog. Neurobiol.

Cholinergic systems in the rat brain. I. Projections to the limbic telencephalon

Brain Res. Bull.

An analysis of the origins of the cholinergic and noncholinergic septal projections to the hippocampal formation of the rat

J. comp. Neurol.

The cholinergic hypothesis of geriatric memory dysfunction

Science

Cited by (54)

Dorsal hippocampal muscarinic cholinergic receptors orchestrate behavioral and autonomic changes induced by contextual fear retrieval

Co-activation of selective nicotinic acetylcholine receptors is required to reverse beta amyloid–induced Ca<sup>2+</sup> hyperexcitation

Cholinergic nervous system and glaucoma: From basic science to clinical applications

Basal Forebrain Cholinergic Circuits and Signaling in Cognition and Cognitive Decline

Cotransmission of acetylcholine and GABA

No evidence for role of extracellular choline-acetyltransferase in generation of gamma oscillations in rat hippocampal slices in vitro