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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nat Neurosci. Author manuscript; available in PMC 2009 July 1.
Published in final edited form as:
Published online 2008 December 21. doi:  10.1038/nn.2238
PMCID: PMC2704296

Olfactory behavior and physiology are disrupted in prion protein knockout mice


The prion protein PrPC is infamous for its role in disease, yet its normal physiological function remains unknown. Here we report a novel behavioral phenotype of PrP−/− mice in an odor-guided task. This phenotype is manifest in three PrP knockout lines on different genetic backgrounds, strong evidence it is specific to the lack of PrPC rather than other genetic factors. PrP−/− mice also display altered behavior in a second olfactory task, suggesting the phenotype is olfactory specific. Furthermore, PrPC deficiency affects oscillatory activity in the deep layers of the main olfactory bulb, as well as dendrodendritic synaptic transmission between olfactory bulb granule and mitral cells. Importantly, both the behavioral and electrophysiological alterations found in PrP−/− mice are rescued by transgenic neuronal-specific expression of PrPC. These data suggest a critical role for PrPC in the normal processing of sensory information by the olfactory system.


Despite two decades of research, the function of the cellular prion protein PrPC is still unknown. It had been hoped the PrP knockout mouse would provide evidence for the function of this protein so widely expressed in all vertebrates, at all stages, and in almost all tissues, especially brain. Such ubiquity suggested PrPC might perform some essential cellular function. However, the first PrP−/− mouse displayed no overt phenotype, implying the protein was dispensable1. Instead, the major finding in PrP−/− mice was their resistance to prion disease2.

Nevertheless, it appears unlikely the PrP protein would have evolved simply to enable a rare fatal disease. Indeed, since the initial knockout mouse study, a host of subtle phenotypes have been described, ranging from behavioral changes to electrophysiological and biochemical alterations3. The reported behavioral phenotypes are of a disparate nature, as might be expected from the widespread expression pattern of PrPC in the brain. They include altered circadian rhythm4, modified sleep patterns5, impaired spatial learning behavior in the Barnes circular maze6, and increased sensitivity to seizure7, 8.

Despite the wide gamut of behaviors tested in PrP knockouts, almost all have relied on spatiovisual or vibrissotactile cues, while to our knowledge olfactory-cued tasks have been overlooked. Since we and others had detected widespread PrPC expression throughout the olfactory system9, 10, we reasoned that olfactory-mediated behaviors might be affected in PrP−/− mice.

The sense of smell is critical to the survival of many animals, mediating such essential behaviors as feeding and mating. The basic circuit of the olfactory system in mice and other mammals, from sensory epithelium to cortex, consists of only two projection synapses (peripheral sensory neuron to mitral cell in the olfactory bulb; mitral cell to pyramidal cell in the cortex) and two layers of inhibitory lateral processing (periglomerular and granule cells) within the olfactory bulb. In particular, mitral and granule cells make a unique dendrodendritic synapse in which mitral cells excite granule cells that reciprocally inhibit the mitral cell. This inhibitory circuit is thought to play a role in synchronizing mitral cell firing and enabling lateral inhibition11, 12.

In our experiments we have uncovered a novel and significant phenotype of PrP−/− mice in the olfactory system by utilizing a combination of genetic, behavioral, and physiological techniques in a systems approach. We employed the so-called “cookie finding task”, a test of broad olfactory acuity, to analyze a battery of mice including PrP knockouts on multiple genetic backgrounds and transgenic mice in which Prnp expression was driven by cell type-specific promoters. In this test, PrP-deficient mice exhibited impaired behavior that was rescued in transgenic mice expressing PrPC specifically in neurons but not in mice expressing only extra-neuronal PrPC. PrP−/− mice displayed altered behavior in an additional olfactory test (habituation-dishabituation) which was also rescued by transgenic neuronal PrP expression, suggesting the phenotype was olfactory specific.

With evidence the underlying alteration resided beyond the periphery, we investigated the odor-evoked electrophysiological properties of the olfactory bulb of PrP knockouts. In these mice, we detected alterations in the patterns of oscillatory activity in the olfactory bulb, and in the plasticity of dendrodendritic synaptic transmission between granule cells and mitral cells. We propose that electrophysiological alterations at the dendrodendritic synapse in the olfactory bulb could underlie the behavioral phenotype we have found.

PrP−/− mice display altered behavior in an olfactory task

We used a test that measures olfactory detection (“cookie finding test”13). Mice that retrieve the cookie faster are thought to have a better sense of smell. The first of two successive trials reflected naïve olfactory-mediated finding; the second the animal's ability to improve based on positive reinforcement received in the first trial.

In Trial 1, wild type (WT) mice retrieved the cookie within a median latency time of 73 s, whereas PrP knockouts (Zürich I line [ZI]; Fig. 1c) were significantly slower at 233 s (p<0.001, Mann-Whitney test; Fig. 1a). Furthermore, close to a third of PrP−/− individuals (6/20) failed to find the cookie within the 10-minute test time, whereas no WT individual failed the test.

Figure 1
Impaired behavior of Zürich I PrP−/− mice in the cookie finding test. (a) Trial 1 of the cookie finding test for B6129 (filled dots) and ZI PrP−/− (open dots). Each dot represents a single individual. Individuals ...

In Trial 2, PrP−/− mice were again significantly slower than WTs at retrieving the cookie (WT median 20 s; PrP−/− median 127.5 s; p<0.001, Mann-Whitney test; Fig. 1b). Even if those PrP−/− animals that had failed Trial 1 were excluded from the analysis based on their failure to have received positive reinforcement, the differences were still significant (WT median 20 s; PrP−/− median 83.5 s; p<0.01 Mann-Whitney test).

The slower latencies of PrP−/− mice in both trials were not due to lack of exploration as assessed by the number of crossings from one cage quadrant to another, nor to lack of appetite since these mice readily consumed the cookie upon finding it, nor to metabolic alteration since all tested mice showed similar weight and daily food consumption regardless of genotype (Supplementary Fig. 1a,b and data not shown). Furthermore, the difference between PrP−/−'s and WTs was not due to locomotor deficiency in the knockouts since both performed similarly in a control version of this experiment where the cookie was presented on the surface of the bedding instead of being buried underneath it (Supplementary Fig. 1c).

Comparing Trial 1 to Trial 2, we observed that WT mice improved from a median of 73 s to 20 s, whereas PrP−/−'s improved at best from 233 s to 83.5 s. 8/9 WT individuals improved between Trials 1 and 2 (lines with negative slopes; Fig. 1d) compared to only 8/13 knockouts (and excluding those that had failed Trial 1 or Trial 2; Fig. 1e). We calculated an improvement factor corresponding to the average ratio of the latency in Trial 1 versus Trial 2. Overall, WTs improved 3-fold (3.12 ± 0.53 SEM) whereas PrP−/−'s only 2-fold (1.97 ± 0.29; p<0.05, one-tailed t test). The difference in degree of improvement was in fact even greater considering the floor effect on WT latencies due to initial rapidity in Trial 1. Thus, PrP−/− mice exhibited impaired behavior in this odor-guided task.

PrP−/− behavior resembles that of anosmic mice

For comparison with a negative extreme of possible behaviors in this task, we tested a known anosmic mouse, the adenylyl cyclase type 3 (AC3) knockout. AC3 is a component of the olfactory transduction cascade necessary for generating action potentials in response to odorant binding at an odorant receptor. AC3−/− mice have been demonstrated to be largely anosmic14 although they retain residual olfactory capacity via their vomeronasal organ15.

To control for the mixed genetic background of both the PrP−/− and AC3−/− mice, we also tested both pure parental strains C57BL/6J (B6) and 129/SvEv (129). Due to animal availability this experiment was conducted in a different facility, necessitating the re-testing of WT B6129 and ZI PrP−/− for comparison to our previous results. The altered environmental conditions may explain the raw data differences for B6129 and PrP−/− mice between the two experiments (Fig. 2 vs Fig. 1).

Figure 2
Zürich I PrP−/− behavior resembles that of a known anosmic, the AC3−/−. (a) Scatter plot showing performance of wild type strains 129, B6, and B6129 (black dots), ZI PrP−/− (open), and AC3−/− ...

Despite these differences, the same trend was apparent under both experimental conditions. All WT mice, regardless of strain, achieved much faster latencies to retrieve the cookie than either the PrP−/− or AC3−/− mice, a significant proportion of which failed both trials (Fig. 2 a,b). In Trial 1, PrP−/− mice, similar to AC3−/− mice, trended towards slower latencies than WTs (Fig. 2a; WT medians: 225.5 s (129); 278 s (B6); 119 s (B6129); PrP−/− median 518 s; AC3−/− median 600 s). In Trial 2, PrP−/−'s continued to resemble AC3−/− mice, failing to improve and contrasting significantly with WTs (Fig. 2b; WT medians: 56 s (129), 79 s (B6), 73 s (B6129 F1); PrP−/− and AC3−/− both 600 s; Fig. 2b).

ZI PrP−/− phenotype extends to other genetic backgrounds

Because the phenotypic impairment had been detected in a PrP−/− mouse on mixed genetic background and lacking wild type littermates, it was possible the phenotype we had detected was due to a genetic factor other than the absence of PrPC. We thus tested two additional PrP−/− lines, one congenic with B6 (Nagasaki; Fig. 3c) and one isogenic with 129 (Edinburgh; Fig. 3l), reasoning if the phenotype were observable on these backgrounds too it might indeed be attributable to PrP deficiency rather than another genetic factor.

Figure 3
The cookie finding phenotype is manifest in PrP knockouts on other genetic backgrounds. (a) Trial 1 of the cookie finding test for B6 (filled) and Nagasaki PrP−/− (open). (b) Trial 2 for mice shown in a. Note the reduced timescale of 5 ...

The Nagasaki (Ng) PrP−/− is not usually a line of choice for phenotypic analysis of PrP deficiency since the mice develop late-onset ataxia due to spurious upregulation of the downstream gene Prnd16. However, below one year of age these mice display no symptoms, and we tested them at the presymptomatic age of 7-10 weeks, much preceding their decline (70 weeks).

We noticed an effect of a predominantly B6 genetic background on cookie finding behavior: Nagasaki PrP−/− mice scored faster latencies than the Zürich I line (Ng median 155 s; ZI median 223s ). In Trial 1, not a single Nagasaki PrP−/− failed to find the cookie versus 6/20 ZI PrP−/− failures (Fig. 3a vs Fig. 1a). Nevertheless, the Nagasaki knockouts were significantly slower than their WT counterparts (PrP+/+ median 76.5 s; PrP−/− median 155 s; p<0.05 Mann-Whitney test; Fig. 3a), thus revealing a phenotype similar to that we had previously detected in the ZI PrP−/− line.

In Trial 2, Nagasaki knockouts were significantly slower than WT counterparts (Fig. 3b; PrP+/+ median 27.5 s; PrP−/− 89.5 s; p<0.001 Mann-Whitney test). The fastest knockout latencies in Trial 2 clustered around 62 s, close to double the median WT latency (Fig. 3b). Although the knockouts tended to improve in Trial 2 (lines with negative slopes, Fig. 3e), they failed to improve as much as WTs (lines with steeper negative slopes, Fig. 3d). Overall, the PrP−/− improvement was almost 2-fold less than WTs (Fig. 3f; PrP+/+ improvement factor 3.84 ± 0.68 SEM; PrP−/− 1.96 ± 0.32; p<0.05, one-tailed unpaired t test). The phenotype exhibited by ZI PrP knockouts was thus confirmed by another knockout line.

However, due to residual 129 alleles tightly linked to the knockout allele in the Nagasaki knockout, which is otherwise congenic with B6, we still could not fully attribute the phenotype to the absence of PrP. We thus tested a third PrP−/−, the Edinburgh line, on a pure 129/Ola background. These mice are isogenic with their WT counterparts, thus circumventing the problem of mixed background (Fig. 3i).

On this background too the phenotype was apparent. Although in Trial 1 PrP+/+ mice only trended towards faster latencies (PrP+/+ median 133.5 s; PrP−/− 227 s; Fig. 3g), they were significantly faster in Trial 2 (PrP+/+ median 26 s; PrP−/− 600 s; p<0.05, Mann-Whitney test; Fig. 3h). In Trial 2, while 4/6 PrP+/+ mice improved to very fast latencies (Fig. 3j), PrP−/− mice showed no clear trend towards improvement, with 4/6 failures (Fig. 3k). The average improvement factors were not significantly different due to the small sample size (PrP+/+ 5.16 ± 2.7; PrP−/− 2.44 ± 2.0; Fig. 3l).

Thus, although the severity of the phenotype varied with the genetic background, we found that on a mixed B6 × 129 background, a congenic B6 background, as well as an isogenic 129/Ola background, PrP−/− mice displayed impaired behavior in the cookie finding test.

The PrP−/− phenotype is rescued by neuronal PrP expression

We next asked whether neuronal-specific PrPC expression could selectively rescue the phenotype. We tested a battery of knockout and transgenic mice all on the Zürich I mixed background (Table 1). We pooled animals according to whether or not they expressed Prnp in neurons, which we confirmed by in situ hybridization, and examined whether neuronal PrPC improved cookie-finding performance. The difference between the two groups (“+neuronal PrPC” and “−neuronal PrPC”) was striking. In both trials, the mice lacking neuronal PrPC were twice as slow as mice that expressed PrPC in neurons (p<0.001, Mann-Whitney test; Fig. 4a,b).

Figure 4
Neuronal PrP expression rescues the cookie finding phenotype. (a). Trial 1 for all lines, neuronal PrPC expressers in black (“+neuronal PrPC”; B6129, Tg20, NSE-PrP); neuronal PrPC-deficient mice in red (“−neuronal PrPC ...
Table 1
Mouse strains tested for cookie finding behavior. Abbreviations: ZI – Zürich I; PrP−/− – PrP knockout; NSE – neuron specific enolase; MBP – myelin basic protein; lck - lymphocyte protein tyrosine ...

This difference was not due to the effect of a single strain, as shown by breaking down the groups into individual data sets (Fig. 4c-d). Overexpression of PrPC on a PrP knockout background (Tg20) exerted a rescuing effect, as did NSE-driven expression of PrPC (i.e. neuronal-specific expression). Both these lines closely resembled the B6129 WT (Fig. 4c,d) with Trial 1 medians all below 100 s and Trial 2 medians below 40 s. In contrast, when PrP was expressed in non-neuronal cells such as myelinating glia (MBP-PrP) or B cells (CD19-PrP), the animals failed to be rescued and phenotypically resembled the ZI PrP−/− (Fig. 4c,d). Additionally, double knockout mice of Prnp and downstream gene Prnd (Prn−/−) were also impaired. All mice lacking neuronal PrPC (open dots) displayed median latencies above 160 s in Trial 1 (Fig. 4c) and above 125 s in Trial 2 (Fig. 4d). The case of Lck-PrP (grey dots, Fig. 4c,d) will be discussed below.

Lck-PrP mice express some neuronal Prnp in the OB

Interestingly, the Lck-PrP transgenic line (grey dots, Figure 4c-d) appeared to be at least partially rescued by its particular pattern of PrP expression. Lck encodes lymphocyte protein tyrosine kinase and is highly expressed in T cells. However, by in situ hybridization we found the Lck promoter to drive Prnp expression in several brain areas, including the OB (juxtaglomerular cells, mitral/tufted cells, and granule cells, Fig. 5a) and the cerebellum (Fig. 5b). In contrast, CD19-PrP mice (B cell-specific expressers) showed no such staining (Fig. 5d). Other reports have also detected an active Lck promoter in neurons of the brain including in olfactory areas17 (Allen Brain Atlas

Figure 5
Lck-PrP transgenic mice express some neuronal PrPC. Fluorescent in situ hybridization for detection of Prnp transcripts in the olfactory bulb and cerebellum of transgenic Lck-PrP mice (a-c) and CD19-PrP mice (d). Left panels show signal from antisense ...

We had thus excluded the Lck-PrP line from the groups shown in Figures 4a,b since in these animals PrPC was expressed in some but not all neurons. However, the substantial rescue mediated by the particular pattern of PrP expression in Lck-PrP mice could in fact point to neurobehavioral regions of importance. In particular, PrPC was not expressed in the olfactory epithelium of these mice (Fig. 5c), suggesting the basis for the impairment was not peripheral. Additionally, we observed normal odor-evoked electro-olfactogram responses from PrP−/− olfactory epithelium (Supplementary Fig. 2). The physiological correlates underlying the impaired behavior thus appeared to reside in central structures. To streamline our investigation, we restricted our subsequent experiments to the use of the B6129 wild type, the ZI PrP−/−, and the NSE-PrP transgenic line (Fig. 4e-f).

Altered behavior of PrP−/− mice in another olfactory task

To help ascertain whether the phenotype of the PrP−/− mice in the cookie finding test was indeed olfactory specific, we performed an additional olfactory behavior test, the habituation-dishabituation assay18. In this test, successive presentations of the same stimulus odor result in a decrease of investigatory behavior (habituation). An increase in the animal's interest when a novel odor is presented (dishabituation) is interpreted as an ability to discriminate the difference between the two odorants. We used a peanut butter (PB) odor as the habituation odor, a mixture of PB and vanilla as the first novel odor, and amyl acetate as an additional, more different, novel odor.

ZI PrP−/− mice habituated to the first odor similar to controls (B6129 and NSE-PrP). However, whereas the controls showed increased interest in the novel odors, PrP−/− mice exhibited altered olfactory behavior by failing to do so (Fig. 4g). Together with the results from the cookie finding test, this result strongly suggested the phenotype was indeed olfactory specific.

Altered physiological responses to odor input in PrP−/− OB

We focused on the electrophysiological properties of the olfactory bulb circuitry because the OB is the first brain area to process olfactory information, and the behaviorally rescued Lck-PrP mice expressed PrPC in neurons of the OB. We recorded local field potentials (LFPs) from this area because they reflect the average current flow from synaptic and spiking activity around the recording site (Fig. 6a). Furthermore, since various frequencies of LFP oscillations specifically reflect different processes, an LFP signal simultaneously assays different types of physiological events. For instance, in anesthetized mice, gamma oscillations (40-100 Hz) reflect activity originating from a specific synapse between output neurons (mitral cells) and interneurons (granule cells) called the dendrodendritic synapse19, 20.

Figure 6
Power of LFPs and duration of the odor response in PrP−/− mice. (a) Local Field Potentials (LFPs) were measured in vivo from the granule cell layer in the main olfactory bulb of anesthetized mice. (b) Example LFP traces from a B6129 mouse ...

In the OB of anesthetized mice, LFP oscillations are coupled to the breathing cycle, allowing us to use breaths as a measure of time (Fig. 6b). We measured the power of LFP oscillations at frequencies ranging from 2 Hz to 120 Hz over a sequence of breaths surrounding odor stimulation. We did not find alterations at beta (10-40 Hz) and delta frequencies (breathing rate, 2-3 Hz; data not shown), so we focused our analysis on gamma (40-70 Hz) and high-gamma (70-100 Hz) oscillations.

During normal (odorless) respiration, PrP−/− animals exhibited significantly lower power than B6129 and NSE-PrP control mice at 88 Hz (Fig. 6c; mean power in 10−3mV2/Hz: PrP−/− 9.01 ± 2.11; B6129 18.80 ± 2.96; NSE-PrP 22.1 ± 5.57; ANOVA p<0.05), 93 Hz (PrP−/− 5.81 ± 1.17 × 10−3mV2/Hz; B6129 17.0 ± 3.05; NSE-PrP 18.0 ± 4.56; ANOVA p<0.05), and 98 Hz (PrP−/− 3.99 ± 0.73 × 10−3mV2/Hz; B6129 12.80 ± 2.32; NSE-PrP 10.50 ± 1.92, ANOVA p<0.05). Similar analysis of the first breath of odor showed an increase in the power of oscillations in gamma and high-gamma compared to odorless respiration, but without any significant differences between the groups (Fig. 6d). Analysis with finer temporal resolution was necessary to resolve any differences (see Fig. 7).

Figure 7
High-frequency oscillations in PrP knockouts are dampened within the course of a single breath. Example waveforms from single mice demonstrating how band power in high-gamma (Γ, 70-100 Hz), gamma (γ, 40-70 Hz), and beta (β, 10-40 ...

Plotting the average band power for every breath allowed us to observe changes in the kinetics of the odor response. Odor stimulation elicited a strong response in both gamma (Fig. 6e) and high-gamma (Fig. 6f) bands, visible as a sharp increase in power followed by a slow decay. In PrP−/−mice, this decay occurred over a significantly larger number of breaths than in either control group for gamma band oscillations (decay time in mean number of breaths: PrP−/− 12.0 ± 1.8; B6129 5.5 ± 1.8; NSE-PrP 5.0 ± 1.7; ANOVA p<0.05; Fig. 6e, right), and high-gamma oscillations (PrP−/− 10.0 ± 1.9; B6129 2.2 ± 0.3; NSE-PrP 2.6 ± 0.8; ANOVA p<0.001; Fig. 6f, right). Together, the lower power yet sustained duration of high-frequency oscillations in the PrP−/− suggested that the temporal structure of oscillations within a single breath might also be altered.

Oscillatory dynamics poorly timed to breathing in PrP−/−

To better understand the oscillatory phenotype, we further analyzed our LFP data to measure the emergence and extinction of LFP oscillations within a breathing cycle. Gamma oscillations in the granule cell layer of the OB emerge during exhalation and are extinguished shortly after (Fig. 7a-c). Surprisingly, the total range of oscillatory power during a breath was smaller in the PrP−/− (Fig. 7b) compared with B6129 and NSE-PrP mice (Fig. 7a,c). Furthermore, the distribution of oscillatory power in the PrP−/− was temporally diffuse across an odor breath (Fig. 7b, right), an alteration that was sustained in a series of breaths following odor exposure (Supplementary Fig. 3).

We quantified the range of oscillatory power within a non-odor and an odor-containing breath (breaths 1 and 5 in Supplementary Fig. 3; Fig. 7), by taking the difference between the peak of power and the following trough. In a non-odor breath, the change in power of high-gamma oscillations in the PrP−/− was reduced compared with B6129 and NSE-PrP mice (Fig. 7d, left; mean ΔPower in dB: PrP−/− 6.5 ± 0.9; B6129 11.5 ± 0.8; NSE-PrP 11.6 ± 1.5). Similarly, in an odor-containing breath, both gamma and high-gamma oscillations exhibited less change in power in PrP−/− mice (Fig 7d, right; PrP−/− 5.9 ± 0.9 dB; B6129 9.7 ± 1.1; NSE-PrP 9.5 ± 0.6).

Oscillations at these high frequency bands (gamma and high-gamma) are believed to result from activity at the dendrodendritic synapse19, 20. The observed alterations in both power and timing thus suggested that the properties of the dendrodendritic synapse may be affected in the PrP−/−.

Altered paired-pulse plasticity of dendrodendritic synapse

We next examined the PrP−/− dendrodendritic synapse for changes that could underlie the observed behavioral phenotypes. We focused on the short-term plasticity of this synapse since our LFP results suggested that PrP−/− mice might have disrupted synchronization between breathing and oscillations, perhaps reflecting altered facilitation or depression of this synapse. We therefore performed paired-pulse stimulation of the synapse by antidromically exciting mitral cells from their axon bundle in the lateral olfactory tract (LOT) (Fig. 8a, top). This stimulation paradigm produces distinct field potentials corresponding to granule cell excitation (field excitatory postsynaptic potentials or fEPSPs) followed by mitral cell inhibition21, 22 (field inhibitory postsynaptic potentials or fIPSPs; Fig. 8a bottom).

Figure 8
Paired pulse synaptic plasticity of field potentials in the GCL after LOT stimulation. (a) Top: Diagram illustrating the stimulation paradigm. MC – mitral cell, GC – granule cell, LOT – lateral olfactory tract. Middle: Example ...

In PrP−/− mice, reciprocal inhibition of mitral cells (fIPSP) showed unusual facilitation over a range of inter-stimulus intervals (Fig. 8c). B6129 and NSE-PrP mice had a significantly facilitated paired pulse ratio from the PrP−/− at intervals between 80 and 100 ms, and B6129's also showed a significantly different ratio at 50 ms (ANOVA with Fisher's PLSD p<0.05). Interestingly, facilitation of the fIPSP in the PrP−/− was not accompanied by any differences in the plasticity of the granule cell fEPSPs (Fig. 8b).


We have thus described a novel olfactory behavioral phenotype of PrP−/− mice, as well as physiological alterations in their olfactory bulb. The cookie finding phenotype was manifest in three PrP−/− lines on alternate genetic backgrounds, strong evidence of its dependence on PrPC rather than other genetic factors. PrP knockouts also displayed altered behavior in the habituation-dishabituation task, suggesting the phenotype was likely olfactory-specific. PrP−/− mice exhibited widespread alterations of oscillatory activity in the OB as well as altered paired-pulse plasticity at the dendrodendritic synapse. Importantly, both the behavioral and electrophysiological phenotypes could be rescued by neuronal PrPC expression. These data suggest a critical role for PrPC in the normal processing of sensory information by the olfactory system.

PrP−/− cookie-finding behavior strikingly resembled that of the anosmic AC3−/−, however PrP−/− mice are clearly not anosmic. Indeed, no aspect of PrP−/− survival suggested they might harbor a deficit in an odor-guided task. Anosmic pups have an 80% neonatal fatality rate due to difficulty suckling at birth and inadequate maternal care, and those that survive have low body weight during their first 3 months14. In contrast, PrP−/−'s have healthy litters of average size (~6-9 pups/litter) that grow to normal body weights. The lack of outward signs of anosmia is likely a reason why olfactory tasks have been overlooked in previous behavioral characterizations of PrP−/− mice.

The behavioral impairment we have detected in PrP knockouts does not originate in the periphery. This is supported by the normal appearance of odor-evoked electro-olfactogram responses from PrP−/− olfactory epithelium, and by the rescued behavior of Lck-PrP mice that do not express PrPC in their olfactory sensory neurons but do in subsets of central neurons, including within the OB. Hence, the PrP−/− behavior deficit likely arises from alterations in central processing events in the OB and/or higher centers.

One initial concern regarding the behavioral phenotype was the mixed background of the Zürich I PrP−/−. Any phenotype of Zürich I knockout mice could be due to genes of 129 origin linked to Prnp rather than to the knockout allele itself. The striking impairments we had detected thus necessitated cautious interpretation.

We confirmed the phenotype of the Zürich I knockouts through two strategies: (1) testing alternate PrP−/− lines with different genetic backgrounds, B6 congenic (Nagasaki) and 129 isogenic (Edinburgh); and (2) using transgenic lines on the Zürich I background that express PrPC in specific cell subtypes.

The use of multiple knockout lines illustrated how genetic background can modulate phenotypic severity. For instance, although the Nagasaki knockouts scored consistently slower latencies than their wild type B6 counterparts, they were on average faster than the ZI knockouts. Furthermore, no Nagasaki individual failed the test whereas a third of ZI knockouts failed each trial. A predominantly B6 background thus appeared to attenuate the phenotype, although no difference was apparent between WT B6 and 129 strains, perhaps due to a floor effect, all WTs achieving an unsurpassable threshold of rapidity.

The transgenic approach demonstrated the phenotype was neuronal-specific. NSE-PrP and Lck-PrP mice were rescued while MBP-PrP and CD19-PrP mice failed to be. Additional tested lines all segregated in a similar fashion, according to whether or not they expressed neuronal PrPC. Perhaps most importantly, the rescued behavior of the NSE-PrP mice proved that the PrP−/− phenotype was indeed due to the absence of PrP and not to genes in the vicinity of Prnp, since the introduction of the PrP transgene alone sufficed to mediate the rescue.

Thus although the phenotype was attributable to lack of PrP, its olfactory specificity remained uncertain due to the behavioral complexity of the cookie finding test. PrP−/− food consumption and body weights appeared no different from controls, allowing us to rule out any possible alterations in metabolism or appetite. Knockouts performed similarly to WTs in a control version of the experiment in which the food stimulus was no longer concealed beneath the bedding. Knockouts were thus fully capable of navigating the test cage and locating the visible cookie, suggesting the deficit in the cookie finding test was thus neither locomotor nor exploratory. Furthermore, PrP−/− mice have been documented to perform normally in tests using extensive locomotor skills such as the Morris water maze1. Importantly, in an additional olfactory assay, PrP−/− mice also exhibited altered behavior, failing to react to a novel odor that was discriminable by NSE-PrP and B6129 mice. Together, the phenotypes in the cookie finding and the habituation-dishabituation tests pointed to an olfactory-specific phenotype.

We thus focused our follow-up investigation on the OB because it contains the initial synapse of the olfactory system and the first circuit to integrate sensory and higher cortical information. We observed disruptions in LFP oscillations and in the plasticity of the dendrodendritic synapse, either, or both, of which could contribute to the PrP−/− behavioral phenotype.

Oscillatory LFPs may act to organize information flow within the olfactory system23, 24 by constraining the timing of mitral cell action potentials25. In addition, gamma oscillations are specifically implicated in behavioral performance in olfactory tasks26-28. Therefore, alterations in oscillatory timing during odor exposure may perturb OB output to higher centers by disrupting how information is packaged within a breathing cycle.

Altering the dendrodendritic synapse may have multiple functional consequences. This synapse may mediate lateral inhibition between ensembles of mitral cells, and be critical for olfactory discrimination29, 30. Additionally, because granule cells receive convergent information onto their proximal dendritic arbor from multiple higher brain areas31, disruption of the dendrodendritic synapse may alter the transmission of centrifugal modulation to OB mitral cells.

High frequency oscillations in the OB (gamma and high-gamma) are shown in vitro to result from the rapid and reciprocal interactions between granule and mitral cells across the dendrodendritic synapse19, 20. Therefore, our data could imply that increased facilitation of the mitral cell IPSP following repetitive spiking decreases the dynamic range and increases the duration of gamma oscillations across the boundaries of a breath. Unfortunately, not enough is currently known about how changes in basic parameters of synaptic physiology manifest themselves on the scale of local field potentials in vivo. Thus, although both oscillatory and synaptic effects could be reversed by neuronal PrPC expression, we cannot claim a causal link between these findings.

Other physiological alterations reported in PrP−/− mice include altered GABA-mediated synaptic currents in CA1 neurons of the hippocampus32 (but see 33), altered long-term potentiation and post-tetanic potentiation34, and altered paired-pulse plasticity within the dentate gyrus6. Given that PrPC is membrane-associated, synaptically enriched35, and present in the external plexiform layer of the OB10, PrPC may function as a member of the synaptic machinery within the OB as well as the hippocampus. Putative molecular partners of PrPC include synaptic molecules such as synapsin Ib36.

We observed that in PrP−/− mice, mitral cells receive facilitated inhibition. This facilitation could result from either pre- and/or post-synaptic changes to the dendrodendritic synapse. Future work should determine the precise synaptic localization of the PrPC protein as well as its biochemical interactions with synaptic machinery. It also remains to be seen whether higher centers involved in olfactory processing and memory are similarly affected by lack of PrPC, or whether analogous synaptic alterations can be detected in other brain regions. Furthermore, the transgenic rescue strategy we used cannot indicate whether the observed phenotypes result from developmental changes in olfactory circuitry. Future use of conditional strategies using tissue specific promoters may allow a more precise dissection of the physiological and behavioral importance of PrPC for olfactory processing.

While the physiological function of PrPC is unknown, its role in the pathogenesis of prion diseases was established beyond reasonable doubt2. The scarcity of any striking pathological phenotypes, particularly in the nervous system, of Prnp-ablated mice was originally taken as evidence that loss-of-function phenomena do not play any role in prion diseases37. The findings reported here suggest that a more nuanced view may be appropriate, and that at least some components of the neurological phenotype of prion infections may be assigned to the malfunction of PrPC-dependent neuronal events.


For fully detailed methods, please refer to the Supplementary methods online.


All PrP-related knockout and transgenic animals shown in Table 1 were provided by Dr. Adriano Aguzzi of the University Hospital of Zürich. Since the Zürich I PrP−/− mice1 are on a mixed C57BL6/J (B6) and 129/SvEv (129) genetic background and lack WT littermates, the F1 hybrid strain of B6 and 129 (B6129) was used as the WT control. AC3−/− mice, also on a mixed B6 and 129 background, were obtained from Dr. Daniel Storm of the University of Washington14. Use of the Edinburgh PrP−/− mice and WT littermates38 was kindly permitted by the IAH (Institute for Animal Health, Compton, Newbury, Berks RG20 7NN, UK) and Dr. Jean Manson of the University of Edinburgh. All animals were housed either at Columbia University or at the University Hospital of Zürich in accordance with institutional requirements for animal care.

Cookie finding behavior test

In this test, a cookie is buried under the cage bedding so as to offer a purely olfactory cue, and the time taken by a mouse to retrieve the cookie is recorded.

Habituation-dishabituation test

The initial interest in an odor presented several times in succession is expected to decrease with each presentation as the animal habituates to the odor. On the 5th presentation, a novel odor is presented. The novelty of the odor should induce an increase in the animal's investigation time, and this is interpreted as an ability to discriminate the difference between odors 1 and 2.

Odor Delivery

A custom-made olfactometer was adapted from a previous design39. Compressed air was humidified and passed by the animal's nose. Odor puffs (2 s) were diverted into the carrier stream. For every mouse, odor was delivered at least 7 times, spaced apart with pulses of solvent headspace.

Electrophysiology recordings

The anesthetized animal's nose was inserted into an air-tight gas mask through which humidified air from the olfactometer was passed. Two craniotomies were performed for insertion of a custom-made tungsten recording electrode40 into the granule cell layer of the MOB, and a custom made bipolar tungsten stimulating electrode into the LOT. Breathing was monitored with a piezoelectric force-transducer (Stoelting, Wood Dale, IL); this signal was used to reliably trigger odor delivery at the transition of inhalation to exhalation (I/E transition).

LFP signal processing and analysis

All signal processing was done off-line using custom written scripts in Spike2, and in Matlab using a combination of custom written scripts and the program eeglab 6.01b42.


Behavior experiments

Statistical analysis was performed using Prism software (GraphPad, San Diego, CA). Cookie finding data were analyzed using non-parametric statistics since the latencies to retrieve the cookie did not follow a normal distribution. The Mann-Whitney test was used for comparison between 2 groups. For comparison between more than 2 groups, we used the Kruskal-Wallis one-way analysis of variance followed by Dunn's post-hoc analysis when a significant overall main effect was found (p<0.05). Habituation-dishabituation data were analyzed using a one-way ANOVA followed by the Bonferroni test when a significant main effect was found (p<0.05).

Physiology experiments

Statistical analysis of physiology data was done using StatView 5.0 (SAS Institute, Cary, NC). Data from three experimental groups was compared using a one-way ANOVA test followed by post-hoc analysis using Fisher's PLSD when a significant overall main effect was found (p<0.05).

Supplementary Material


The authors thank members of the Aguzzi laboratory at the University Hospital of Zürich for their assistance especially Dr. Gino Miele and Petra Schwartz. We also thank Dr. Jean Manson of the University of Edinburgh for the use of the Edinburgh PrP−/− line, Dr. DongJing Zou and Dr. Darcy Kelley of Columbia University for helpful comments on the behavioral experiments, and Dr. Joshua Gordon of Columbia University for valuable discussions on the electrophysiology data. This work was supported by grants from the National Institute on Deafness and Other Communication Disorders (S.F., C.L.P., M.T.V., B.T.S., and A.T.C.). C.L.P. also received a Short Term Fellowship from the European Molecular Biology Organization. A.A. and M.P. were supported by grants from the European Community and the Swiss National Science Foundation.


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