Medial auditory thalamic input to the lateral pontine nuclei is necessary for auditory eyeblink conditioning

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Abstract

Auditory and visual conditioned stimulus (CS) pathways for eyeblink conditioning were investigated with reversible inactivation of the medial (MPN) or lateral (LPN) pontine nuclei. In Experiment 1, Long-Evans rats were given three phases of eyeblink conditioning. Phase 1 consisted of three training sessions with electrical stimulation of the medial auditory thalamic nuclei (MATN) paired with a periorbital shock unconditioned stimulus (US). An additional session was given with a muscimol (0.5 μL, 10 mM) or saline infusion targeting the LPN followed by a recovery session with no infusions. The same training and testing sequence was then repeated with either a tone or light CS in phases 2 and 3 (counterbalanced). Experiment 2 consisted of the same training as Experiment 1 except that muscimol or saline was infused in the MPN during the retention tests. Muscimol infusions targeting the LPN severely impaired retention of eyeblink conditioned responses (CRs) to the MATN stimulation and tone CSs but only partially reduced CR percentage to the light CS. Muscimol infusions that targeted the MPN had a larger effect on CR retention to the light CS relative to MATN stimulation or tone CSs. The results provide evidence that the auditory CS pathway necessary for delay eyeblink conditioning includes the MATN-LPN projection and the visual CS pathway includes the MPN.

Introduction

Pavlovian eyeblink conditioning has been used with great success to delineate the neural mechanisms underlying associative learning and memory (Christian and Thompson, 2003, Thompson, 2005). Eyeblink conditioning is typically established by pairing a tone or light conditioned stimulus (CS) with an unconditioned stimulus (US) that elicits the eyeblink reflex. An eyeblink conditioned response (CR) emerges during paired training that precedes the onset of the US. The intermediate cerebellum, specifically the interpositus nucleus and cerebellar cortex, has been implicated as the site of learning-related plasticity necessary for the eyeblink CR (Freeman et al., 2005, Garcia and Mauk, 1998, Jirenhed et al., 2007, Krupa and Thompson, 1997, Krupa et al., 1993, McCormick et al., 1982, McCormick and Thompson, 1984a, McCormick and Thompson, 1984b, Nicholson and Freeman, 2002). Convergence of CS and US information in the cerebellum is necessary for establishing this learning-related plasticity (Thompson, 1976). The dorsal accessory inferior olive provides the cerebellum with US input through climbing fibers in the inferior cerebellar peduncle that synapse on neurons in the interpositus nucleus and Purkinje cells in the cortex (Brodal, 1981, Ito, 1984, Mauk et al., 1986, McCormick et al., 1985, Mintz et al., 1994, Sugihara et al., 2001). The pontine nuclei (PN) provide the necessary and sufficient mossy fiber input to the cerebellum for eyeblink conditioning with auditory, somatosensory, and visual CSs (Bao et al., 2000, Campolattaro and Freeman, 2008, Freeman and Rabinak, 2004, Freeman et al., 2005, Hesslow et al., 1999, Knowlton and Thompson, 1988, Lewis et al., 1987, Steinmetz et al., 1986, Steinmetz and Sengelaub, 1992, Steinmetz et al., 1987, Tracy et al., 1998). The PN show learning-related changes in activity that depend on feedback from the cerebellar nuclei and red nucleus (Cartford et al., 1997, Clark et al., 1997). Thus, pontine–cerebellar interactions may influence eyeblink conditioning.

The neural pathways for different modality CSs may be segregated within the PN. Bilateral electrolytic lesions of the lateral and dorsolateral pontine nuclei (LPN) impair CR retention to a tone CS, but have no effect on conditioning to a light CS in rabbits (Steinmetz et al., 1987). Click-evoked field potentials were also stronger in the lateral and dorsolateral PN relative to the medial pontine nuclei (MPN; Steinmetz et al., 1987). The findings of the Steinmetz et al. study suggest that CS inputs to the cerebellum are anatomically segregated at the level of the PN in rabbits. However, tone evoked neuronal activity is evident throughout the basilar pons in rats (Freeman & Muckler, 2003). Axonal projections from auditory and visual areas also show a high degree of overlap in the rat PN (Campolattaro et al., 2007, Graybiel, 1974). It is therefore possible that the auditory and visual pathways within the PN are not segregated in rats.

Stimulation of auditory structures can support eyeblink CRs, and many of these structures have a direct unilateral projection to the PN that may be important for eyeblink conditioning. The auditory cortex, cochlear nucleus (CN), inferior colliculus (IC), and medial auditory thalamic nuclei (MATN) all have direct projections to the PN (Campolattaro et al., 2007, Kawamura, 1975, Knowlton et al., 1993, Steinmetz et al., 1987). Electrical stimulation of these auditory structures can be used as a CS to support acquisition of eyeblink conditioning (Campolattaro et al., 2007, Knowlton and Thompson, 1992, Nowak et al., 1999, Patterson, 1971). The direct PN projections from the auditory cortex are not necessary as decortication does not block CR acquisition or retention (Oakley and Russell, 1972, Oakley and Russell, 1977). In contrast, lesions of the MATN contralateral to the conditioned eye in rats completely block acquisition and retention of auditory eyeblink CRs, but do not prevent acquisition to a light CS, indicating that the MATN projection to the PN may be critical for eyeblink conditioning with an auditory CS (Halverson and Freeman, 2006, Halverson et al., 2008). The current study was designed to determine whether the MATN-PN projection is necessary for auditory eyeblink conditioning in rats. Anatomical segregation of auditory and visual CS pathways within the PN was also examined.

Section snippets

Experiment 1

Experiment 1 was designed to determine whether the MATN projection to the LPN is necessary for auditory eyeblink conditioning in rats. Electrical stimulation of the MATN was used as a CS (Campolattaro et al., 2007) in the first phase of training. After three sessions of acquisition training with the stimulation CS, muscimol or saline was infused into the LPN during a retention test, followed by a recovery session with no infusions. The rats were then given the same sequence of training and

Experiment 2

Experiment 2 examined whether inactivation of the MPN would produce differential effects on retention of CRs elicited by MATN stimulation, tone, or light CSs. Electrolytic lesions of the middle cerebellar peduncle, which is the primary pontine mossy fiber input into the cerebellum, block retention of light evoked CRs, indicating the pontine nuclei relay visual CS information into the cerebellum for eyeblink conditioning as well as tone CS information (Lewis et al., 1987). Neural tracing studies

Combined retention test analyses

Additional analyses were run to determine possible differences in CR retention between rats receiving muscimol or saline infusions into the LPN or MPN in Experiments 1 and 2. Repeated measures ANOVA on the CR percentage data for the muscimol retention tests, the sessions prior to the muscimol tests, and the sessions after the muscimol tests (sessions 3–5, 8–10, and 13–15) (Fig. 3, Fig. 6) for the LPN, MPN, and saline groups revealed an interaction of the stimulus, session, and group factors F

Discussion

Acquisition of delay eyeblink conditioning with unilateral MATN stimulation as the CS was rapid, as previously reported (Campolattaro et al., 2007). Savings was observed when the CS was switched from MATN stimulation to a peripheral CS relative to de novo acquisition with a peripheral CS (Campolattaro & Freeman, 2009). Retention of eyeblink conditioning with MATN stimulation, tone, or light CSs was severely impaired by muscimol inactivation of the PN contralateral to the conditioned eye. There

Acknowledgment

This research was supported by National Institute for Mental Health Grant MH080005 to J.H.F.

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