Comparative ontogenic profile of cholinergic markers, including nicotinic and muscarinic receptors, in the rat brain

Abstract

The ontogenic profiles of several cholinergic markers were assessed in the rat brain by using quantitative in vitro receptor autoradiography. Brain sections from animals at different stages of development were processed with [3H]AH5183 (vesamicol; vesicular acetylcholine transport sites), [3H]N-methylcarbamylcholine (alpha(4)beta(2) nicotinic receptor sites), [3H]hemicholinium-3 (high-affinity choline uptake sites), [3H]3-quinuclidinyl benzilate (total population of muscarinic receptor sites), [3H]4-DAMP (muscarinic M1/M3 receptor sites), [3H]pirenzepine (muscarinic M1 receptor sites), and [3H]AF-DX 116 and [3H]AF-DX 384 (muscarinic M2 receptor sites) as radiolabeled probes. The results revealed that, by the end of the prenatal period (embryonic day 20), the densities of nicotinic receptor and vesicular acetylcholine transport sites already represented a considerable proportion of those observed in adulthood (postnatal day 60) in different laminae of the frontal, parietal, and occipital cortices, in the layers of Ammon's horn fields and the dentate gyrus of the hippocampal formation, as well as in the amygdaloid body, the olfactory tubercle, and the striatum. In contrast, at that stage, the densities of total muscarinic, M1/M3, M1, and possibly M2 receptor and high-affinity choline uptake sites represent only a small proportion of levels seen in the adult. Differences were also observed in the postnatal ontogenic profiles of nicotinic, muscarinic, vesamicol, and high-affinity choline uptake sites. For example, between postnatal weeks 3 and 5, the levels of M1/M3 and M1 sites were at least as high as in the adult, whereas M2 and high-affinity choline uptake site densities appeared to be delayed and to reach adult values only after postnatal week 5. With regard to cholinergic innervation in the developing rat brain, the present findings suggest a temporal establishment of several components of the cholinergic systems. The first components are the vesicular acetylcholine transporter and nicotinic sites; these are followed by M1/M3 and M1 sites and, finally, by M2 and high-affinity choline uptake sites.

Short-Term Adolescent Nicotine Exposure has Immediate and Persistent Effects on Cholinergic Systems: Critical Periods, Patterns of Exposure, Dose Thresholds

“…Although direct evidence for adolescent brain cell injury caused by low-dose nicotine exposure is being pursued in a separate study (AbreuVillaça et al, 2003), the current results for ChAT provide some evidence of neurotoxicant actions. ChAT is a constitutive cholinergic synaptic biomarker, so that its activity largely reflects the density of cholinergic innervation (Aubert et al, 1996;Happe and Murrin, 1992;Navarro et al, 1989;Slotkin et al, 1990;Zahalka et al, 1992Zahalka et al, , 1993. In our earlier work with a 2-week infusion of 6 mg/kg/day of nicotine, we found small, but statistically significant decrements in ChAT (Trauth et al, 2000a) that corresponded to loss of neural cells (Trauth et al, 2000b).…”

“…Accordingly, ChAT increases during cholinergic synaptogenesis but does not change in response to stimuli that alter cholinergic neuronal activity (Aubert et al, 1996;Happe and Murrin, 1992;Navarro et al, 1989;Slotkin et al, 1990;Zahalka et al, 1992Zahalka et al, , 1993. In contrast, high-affinity choline uptake, as assessed with the binding of HC-3 to the presynaptic high-affinity choline transporter, is responsive to neuronal activity (Klemm and Kuhar, 1979;Simon et al, 1976), and the comparative changes in ChAT and HC-3 binding or transporter function permit distinction between effects on synaptic outgrowth as distinct from synaptic activity (Aubert et al, 1996;Happe and Murrin, 1992;Navarro et al, 1989;Slotkin et al, 1990;Zahalka et al, 1992Zahalka et al, , 1993. Both ChAT and HC-3 binding have been used previously to characterize the differences between adolescent and adult responses to nicotine (Slotkin, 2002;Trauth et al, 2000a).…”

Prenatal Nicotine Exposure Alters the Response to Nicotine Administration in Adolescence: Effects on Cholinergic Systems During Exposure and Withdrawal

“…Nicotine itself does not directly alter ChAT expression or enzymatic activity, nor does nicotine treatment in adulthood affect ChAT (Lapchak et al, 1989;Wessler et al, 2003). Furthermore, as a constitutive biomarker for cholinergic synapses, ChAT largely reflects the concentration of cholinergic terminals and is insensitive to changes in neural impulse activity (Aubert et al, 1996;Happe and Murrin, 1992;Navarro et al, 1989;Slotkin et al, 1990;Zahalka et al, 1992Zahalka et al, , 1993. Accordingly, our results suggest that prenatal nicotine exposure elicits a small but significant increase in the density of cholinergic innervation, likely in reaction to adverse effects on neuronal size and the relative thickness of the neuropil that are detectable in the period immediately preceding and during the increase in ChAT Sabherwal, 1994, 1998;Roy et al, 2002).…”

“…ChAT, the enzyme responsible for acetylcholine biosynthesis, is a constitutive marker for cholinergic nerve terminals and serves as an archetypal measure of cholinergic innervation, but its activity does not respond to changes in impulse flow. Accordingly, ChAT reflects the concentration of cholinergic nerve terminals but does not change in response to stimuli that alter cholinergic neuronal activity (Aubert et al, 1996;Happe and Murrin, 1992;Navarro et al, 1989;Slotkin et al, 1990;Zahalka et al, 1992Zahalka et al, , 1993. In contrast, high affinity choline uptake, as assessed with the binding of HC-3 to the presynaptic high-affinity choline transporter, is responsive to neuronal activity (Klemm and Kuhar, 1979;Simon et al, 1976) and the comparative changes in ChAT and HC-3 binding or transporter function (HC-3/ChAT ratio) permit distinction between effects on the concentration of synaptic terminals as distinct from those on synaptic activity (Aubert et al, 1996;Bissette et al, 1996;Happe and Murrin, 1992;Navarro et al, 1989;Slotkin et al, 1990;Zahalka et al, 1992Zahalka et al, , 1993.…”

Unconventional ligands and modulators of nicotinic receptors

“…This is particularly relevant because ␣7 nAChRs seem to play a significant role in numerous aspects of development of cholinergic synapses both at the neuromuscular junction (Romano et al, 1997;Fischer et al, 1999) and in the nervous systems (Broide and Leslie, 1999). It is possible that during maturation of the nervous systems choline acts as the primary endogenous agonist of ␣7 nAChRs, given that expression of the ACh synthesizing enzyme choline acetyltransferase lags behind the appearance of nAChRs in developing neurons (Chiappinelli and Giacobini, 1978;Yamada et al, 1986;Aubert et al, 1996). In addition, taking into account the ubiquitous distribution of cholinesterases in the CNS, it is unlikely that high concentrations of ACh will reach ␣7 nAChRs away from transmitter release sites.…”