Ventral lateral geniculate input to the medial pons is necessary for visual eyeblink conditioning in rats

The conditioned stimulus (CS) pathway that is necessary for visual delay eyeblink conditioning was investigated in the current study. Rats were initially given eyeblink conditioning with stimulation of the ventral nucleus of the lateral geniculate (LGNv) as the CS followed by conditioning with light and tone CSs in separate training phases. Muscimol was infused into the medial pontine nuclei (MPN) after each training phase to examine conditioned response (CR) retention to each CS. The spread of muscimol infusions targeting the MPN was examined with fluorescent muscimol. Muscimol infusions into the MPN resulted in a severe impairment in retention of CRs with the LGNv stimulation and light CSs. A less severe impairment was observed with the tone CS. The results suggest that CS information from the LGNv and light CSs is relayed to the cerebellum through the MPN. Retrograde tracing with fluoro-gold (FG) showed that the LGNv and nucleus of the optic tract have ipsilateral projections to the MPN. Unilateral inputs to the MPN from the LGNv and nucleus of the optic tract may be part of the visual CS pathway that is necessary for visual eyeblink conditioning.The neural substrates of associative motor learning have been studied extensively using eyeblink conditioning (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 over the course of paired training, and the peak of eyelid closure occurs at the onset time of the US. Results from experiments using temporary lesions of the cerebellar deep nuclei or cerebellar cortex indicate that the anterior interpositus nucleus and cerebellar cortex are necessary for acquisition, expression, and extinction of eyeblink conditioning (Krupa et al. 1993; Hardiman et al. 1996; Krupa and Thompson 1997; Garcia and Mauk 1998; Medina et al. 2001; Bao et al. 2002; Freeman et al. 2005a). Blocking cerebellar output with inactivation of the superior cerebellar peduncle, red nucleus, or brainstem motor nuclei selectively blocks CR expression but not acquisition, providing further evidence that learning occurs in the cerebellum (Chapman et al. 1990; Krupa et al. 1993, 1996; Krupa and Thompson 1995).Sensory stimuli from every modality are sent to the pontine nuclei (PN), which receive projections from the lower brainstem, thalamus, and cerebral cortex (Glickstein et al. 1980; Brodal 1981; Mihailoff et al. 1989; Schmahmann and Pandya 1989; Wells et al. 1989; Knowlton et al. 1993; Campolattaro et al. 2007). Neurons in the PN project CS information to the cerebellum via mossy fibers in the middle cerebellar peduncle that synapse on granule cells in the cerebellar cortex and on neurons in the interpositus nucleus (Bloedel and Courville 1981; Brodal 1981; Steinmetz and Sengelaub 1992; Mihailoff 1993). Lesions of the middle cerebellar peduncle impair eyeblink conditioning with auditory, somatosensory, and visual CSs (Lewis et al. 1987). Bilateral electrolytic lesions of the dorsolateral and lateral pontine nuclei (LPN) block retention of CRs to an auditory CS but have no effect on light-elicited CRs (Steinmetz et al. 1987). Inactivation of the contralateral LPN blocks CRs to a tone CS but not to lateral reticular nucleus stimulation in rabbits (Bao et al. 2000). Moreover, stimulation of the LPN or middle cerebellar peduncle is a sufficient CS for eyeblink conditioning (Steinmetz et al. 1986, 1987; Tracy et al. 1998; Bao et al. 2000; Freeman and Rabinak 2004; Freeman et al. 2005b; Campolattaro and Freeman 2008). The findings from the lesion, inactivation, and stimulation studies provide evidence that the PN is the proximal part of the CS pathway for cerebellar learning. These studies also indicate that the LPN is the primary source of auditory CS input to the cerebellum.Only a few studies have examined the visual CS pathway necessary for eyeblink conditioning. The dorsal and ventral divisions of the lateral geniculate nucleus of the thalamus (LGNd, LGNv), pretectal nuclei, visual cortex (VCTX), and superior colliculus (SC) comprise a hypothesized parallel visual CS pathway for eyeblink conditioning (Koutalidis et al. 1988). Combined lesions of all of these visual areas completely block acquisition, lesions of two visual areas produce a partial impairment, and lesions in one visual area do not impair CR acquisition (Koutalidis et al. 1988). Stimulation of the VCTX, SC, and LGNv support eyeblink conditioning, and each of these structures has a direct unilateral projection to the PN that could be important for eyeblink conditioning (Halverson et al. 2009). The lesion and stimulation studies provide evidence that structures in the hypothesized visual CS pathway are independently capable of supporting conditioning. An important aspect of the visual CS pathway proposed in the Koutalidis et al. (1988) study is distributed projections of each visual area to different regions of the PN. The important projections were hypothesized to be from the VCTX to the rostral portion of the PN, from both the SC and pretectal nuclei to the dorsolateral PN, and the LGNv projection to the medial pontine nuclei (MPN) (Koutalidis et al. 1988). Lesions of the VCTX were substituted for LGN lesions in the Koutalidis et al. (1988) study due to technical problems with animal survival. The LGNv projection to the MPN was therefore not examined in their combined lesion group. Stimulation of the anterior pretectal nucleus is not a sufficient CS to support eyeblink conditioning (Campolattaro et al. 2007). The direct PN projection from the VCTX is not necessary for CR retention to a light CS, as lesions do not prevent eyeblink conditioning to a light CS in dogs or monkeys (Hilgard and Marquis 1935, 1936). Moreover, lesions of the entire cerebral cortex do not prevent acquisition or retention of delay eyeblink conditioning to a tone or light CS in rabbits (Oakley and Russell 1972, 1977). The LGNv and SC, therefore, are likely sources of visual input to the PN that is necessary for eyeblink conditioning.The current experiment investigated whether information from the LGNv and a visual CS (light) share similar inputs into the MPN and whether those inputs are different from an auditory CS. The visual projections to the MPN were also investigated with the retrograde tracer fluoro-gold (FG) to identify structures that may be involved with the relay of CS information during eyeblink conditioning. In the conditioning experiment, rats received three phases of training, with each phase consisting of three acquisition sessions followed by a muscimol infusion into the MPN, and then a saline recovery session. Each rat received unilateral stimulation of the LGNv (contralateral to the trained eye) during phase 1 of training followed by either a tone or light CS in phases 2 and 3 (order of stimulus presentation was counterbalanced). One group received LGNv stimulation in phase 1 followed by a light CS in phase 2, and a tone CS in phase 3 (SLT). The other group received the tone CS in phase 2, and light CS in phase 3 (STL).