Genetically induced cholinergic hyper-innervation enhances taste learning

Abstract

Acute inhibition of acetylcholine (ACh) has been shown to impair many forms of simple learning, and notably conditioned taste aversion (CTA). The most adhered-to theory that has emerged as a result of this work - that ACh increases a taste's perceived novelty, and thereby its associability - would be further strengthened by evidence showing that enhanced cholinergic function improves learning above normal levels. Experimental testing of this corollary hypothesis has been limited, however, by side-effects of pharmacological ACh agonism and by the absence of a model that achieves long-term increases in cholinergic signaling. Here, we present this further test of the ACh hypothesis, making use of mice lacking the p75 pan-neurotrophin receptor gene, which show a resultant over-abundance of cholinergic neurons in sub-regions of the basal forebrain (BF). We first demonstrate that the p75-/- abnormality directly affects portions of the CTA circuit, locating mouse gustatory cortex (GC) using a functional assay and then using immunohistochemisty to demonstrate cholinergic hyper-innervation of GC in the mutant mice - hyper-innervation that is unaccompanied by changes in cell numbers or compensatory changes in muscarinic receptor densities. We then demonstrate that both p75-/- and wild-type (WT) mice learn robust CTAs, which extinguish more slowly in the mutants. Further testing to distinguish effects on learning from alterations in memory retention demonstrate that p75-/- mice do in fact learn stronger CTAs than WT mice. These data provide novel evidence for the hypothesis linking ACh and taste learning.

Keywords: cholinergic system; conditioned taste aversion; p75 knockout mouse; taste learning.

Figure 1

Gustatory cortex in the mouse. The left hemisphere of this figure is a schematic diagram of the likely coronal plane of mouse (reprinted with permission from Paxinos and Franklin, 2001) gustatory cortex (GC), selected for its homology to the known location of rat GC (Katz et al., 2001). The super-imposed squares and circles show the location of cannula tips for muscimol and control mice, respectively, in the GC localization experiments – most tips are found in granular (GI) or dysgranular (DI) insular cortex; also noted are dorsal and ventral agranular insular (AID and AIV) as well as piriform cortex (Pir). The right hemisphere shows a photomicrograph from one mouse subject (with the approximate demarcations of granular, dysgranular, and agranular cortex noted). The cannula track is readily visible, as is the localized spread of fluorescent muscimol infused through that cannula just before perfusion. Scale bar = 500 μm.

Figure 2

Functional test of mouse GC localization. (A) A muscimol concentration that had relatively little impact on ad lib consumption (i.e., 0.50 μg/μl), infused into putative GC just before a CTA training session (a single pairing of orally administered NaCl and ip injected LiCl), inhibited taste learning – these mice did not reduce their NaCl consumption in the testing session (y-axis). Mice receiving control (saline) infusions into putative GC learned normally, consuming much less NaCl after training, as did controls receiving muscimol infusions into non-GC sites. Thus we can conclude that the infusion cannulae have correctly targeted mouse GC. (B) In a parasagittal view, the locations, and approximate spreads, of muscimol infusions that were most effective at blocking CTA. Error bars, here and in every figure, represent the standard error of the mean, ** = p < 0.01; see text for further details. S1, somatosensory cortex; GI, DI, AID, AIV, gustatory insular cortex (granular, dysgranular, agranular dorsal and ventral); Pir, piriform cortex; CPU, caudate putamen.

Figure 3

p75−/− mice showed cholinergic hyper-innervation in GC compared to wild-type mice. (A) The two photomicrographs show ChAT-stained sections through GC. More fibers are visible in p75−/− mice than in wild-type. The group data are summarized in the panel to the right, which shows that in the p75−/− mouse, a higher percentage of the space in the images (y-axis) was taken up by ChAT-stained fibers than in the wild-type (WT) mouse. (B) The same analysis done on skeletonized images (see Materials and Methods for details), which allows us to rule out confounding explanations having to do with the possibility of brighter staining. The group data demonstrates that, when stained fibers are reduced to equi-luminant, equi-thick lines, these cholinergic fibers are longer in p75−/− mice than in wild-type mice. ** = p < 0.01, scale bar = 100 μm.

Figure 4

p75−/− and wild-type mice are comparable in cortical neuron number and cholinergic innervation of gustatory thalamus. (A) The photomicrographs show NeuN staining, which labels all neurons in wild-type and p75−/− GC slices. The group data at right demonstrates that the two strains did not differ in the number of neurons in GC, indicating that cholinergic hyper-innervation did not change the number of cortical neurons. (B) Like Figure 3B, this figure shows ChAT staining of slices harvested from p75−/− and wild-type mice – this time from gustatory thalamus, which receives cholinergic innervation from brainstem rather than basal forebrain. The photomicrographs (left) and group data (right) reveal no major p75/wild-type differences in cholinergic innervation of this important part of the mouse gustatory system. Scale bar = 100 μm.

Figure 5

p75−/− and wild-type GC differed in expression of the M2, but not M1 or M4, cholinergic receptor mRNAs. RNA was isolated from GC of wild-type and p75−/− mice and the levels of M1, M2, and M4 receptor mRNA were measured relative to expression of GAPDH using real-time PCR. Levels of mRNA are expressed relative to GAPDH mRNA expression levels, showing no change in M1 and M4 levels and a decrease in M2 mRNA. * = p < 0.05.

Figure 6

p75−/− mice learn stronger CTAs than wild-type mice. (A) With the standard dose of LiCl (0.15 M, 2% of body weight), both wild-type (open ovals), and p75−/− mice (closed triangles) learn strong CTAs, reducing their consumption of saccharin in the first testing session to ~20% of training-session consumption (y-axis). Across further testing sessions (x-axis) both groups re-learned to consume saccharin (i.e., the CTA underwent extinction), but this occurred faster for wild-type mice (see text for statistics). (B) When the LiCl dose was reduced to 1% of body weight, the learning was accordingly milder. By lifting consumption away from the floor, it became possible to observe a difference in initial learning: p75 mice learned stronger CTAs (i.e., consumed less saccharin) than wild-type mice. This difference was maintained through several extinction trials. See text for statistical details.