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The interposed nuclei (IN) of the cerebellum play a crucial role in the classically conditioned eyeblink circuit. It has previously been shown in well trained animals that injecting the IN with GABAA antagonists produces short latency conditioned responses (SLRs). The mechanism underlying SLR generation is not clear. According to one concept, SLRs originate in cerebellar nuclei in response to direct inputs from collaterals of mossy fibers. An alternate explanation is that SLRs are produced by extra-cerebellar circuits that are excited by increased tonic activity in cerebellar nuclei or by the combined action of inputs to cerebellar nuclei from mossy fiber collaterals and incompletely blocked Purkinje cells. In the present study, we examined whether cerebellar afferent axons in the middle cerebellar peduncle (MCP) participate in SLR expression. We hypothesized that if SLRs are evoked by the sensory mossy fiber input to the IN and cerebellar cortex, then blocking the MCP should abolish these responses. Well trained animals, which had been implanted with dual injection cannulae in the left IN and the left MCP, were injected with gabazine (GZ) into the IN to produce SLRs followed by an injection of the sodium channel blocker tetrodotoxin (TTX) into the MCP. TTX infusions in the MCP suppressed both CRs and SLRs. These findings suggest that the expression of SLRs depends on both direct and cerebellar cortex-mediated sensory information from the mossy fiber system.
The cerebellar interposed nuclei (IN) are a pivotal component in the eyeblink conditioning circuit. The IN receives two major inputs. The first is a massive GABAergic projection from Purkinje cells of the cerebellar cortex (Ito, 1984). The second and appreciably weaker input is from presumably glutamatergic collaterals of cerebellar afferent mossy and climbing fibers (Cicirata et al., 2005; Voogd, 1995). Important insights into the operation of eyeblink conditioning networks were gained in studies manipulating the GABAergic IN input. It has been shown that activating cerebellar cortical projections to the cerebellar nuclei by injecting GABAA agonists into the IN prevents the acquisition and expression of eyeblink conditioned responses (CRs) (Krupa et al., 1993; Bracha et al., 1994; Hardiman et al., 1996). On the other hand, blocking GABAA neurotransmission in the IN dramatically increases the spontaneous firing rate of IN neurons and suppresses their task-related responses (Aksenov et al., 2004). This physiological effect is associated with the abolition of CRs (Mamounas et al., 1987; Attwell et al., 2002; Aksenov et al., 2004). An incomplete disruption of GABAA neurotransmission elevates the IN neuronal firing rate, but it does not eliminate task-related neuronal responses (Aksenov et al., 2004). This physiological state alters CR expression by shortening their latencies and by modifying their temporal profile (Garcia and Mauk, 1998). The neurophysiological mechanisms of these short-latency CRs (SLRs) are not clear. According to one concept, SLRs could be triggered by direct inputs from collaterals of mossy fibers to cerebellar nuclei. Ohyama and colleagues (2006) examined this proposal by blocking direct mossy fiber inputs by infusing the IN of rabbits generating SLRs with blockers of fast glutamate receptors. This treatment reduced the amplitude of SLRs, but it did not eliminate CRs. The persistence of CRs following blocks of glutamate- and GABA-mediated IN inputs in these experiments seems to suggest that the residual CRs were generated by extra-cerebellar circuits. This suggestion, however, is inconclusive because the SLR-inducing GABAA blockade is most likely incomplete (Aksenov et al., 2004; Parker et al., 2009) and therefore conditioned stimulus (CS) signals could still enter the IN via the cerebellar cortex.
In the present study, we examined the cerebellar dependency of SLRs by blocking both direct CS input to the IN and CS input to the cerebellar cortex. This was achieved by inactivating the middle cerebellar peduncle (MCP). The MCP is a fiber tract originating in the pontine nuclei and it relays information encoding the CS to the cerebellum. The MCP projects directly to the IN via mossy fiber collaterals, but its main target is the cerebellar cortex which processes the CS and the unconditioned stimulus (US) information and conveys the results to the IN. We hypothesized that if SLRs are driven by cerebellum-mediated CS signals, then blocking the MCP should abolish these responses. Well trained animals, which had been implanted with dual-injection cannulae in the left IN and the left MCP, were injected with the GABAA antagonist gabazine (GZ) into the IN to produce SLRs, and subsequently the sodium channel blocker tetrodotoxin (TTX) was injected into the MCP to block mossy fiber inputs to the cerebellum. Here we report that blocking the MCP prevented the expression of both CRs and SLRs.
Injections of GZ in the IN shortened CR latencies. In the absence of this GZ action, administering TTX in the MCP suppressed CR expression. Injections of TTX in the MCP of animals exhibiting GZ-induced SLRs suppressed all responses to the CS. Similar to our previous observations (Parker et al., 2009), GZ injections also increased tonic eyelid closure. All of these effects were observed at injection sites located directly at or in the near vicinity of the left anterior IN (Fig. 1) and the left MCP (Fig. 2).
Injections of GZ in the IN were administered in well trained animals at the location determined during the mapping phase of the experiment. The effective dose of GZ differed among animals, varying from 0.125 – 4.11 nmol, and it was carefully chosen to shorten CR latency (Fig. 3A–B). On the other hand, larger doses of GZ abolished CRs (not shown). When compared to control injections of aCSF, infusions of SLR-inducing doses of GZ significantly shortened CR latency (Fig. 4B, F9,27 = 4.17, p = 0.0019) without affecting CR incidence in all four animals (Fig. 4A). The onset of GZ effects on CR latency varied between rabbits, and at the group level this was manifested as a gradual decline in CR latency during the first 3 blocks of the post-GZ period (Fig. 4B). Prior to the injection, CR latency was 158.23 ± 13.87 ms, becoming shortest in post-injection block 5 with an average of 65.47 ± 13.08 ms.
The MCP is a pathway that is understood to supply the cerebellum with the sensory CS signal. Consistent with this concept, inactivating the MCP with TTX severely suppressed CRs (Fig. 3C). Typically, TTX-injected animals ceased responding to the CS with an infrequent exception of isolated CRs or CS-triggered eyelid opening. When compared to control injections of PBS, micro-injections of TTX (3.13 – 9.39 pmol) significantly suppressed CR incidence in all four rabbits (Fig. 4C, F9,27 = 6.85, p = 0.000001). Prior to the injection, animals expressed 100 % CRs. Following TTX, CR incidence quickly declined to less than 15 % and despite a slight increase during post-injection blocks 6–8, it remained depressed throughout the experiment (Fig. 4C). As a consequence of CR suppression, eyeblink latency increased TTX injections (Fig. 4D, F9,27 = 10.66, p = 0.000001). Before the injection, the mean eyeblink latency was 124.67 ± 14.27 ms, and it lengthened to a mean above 350 ms for the majority of post-injection trials (Fig. 4D).
TTX is a sodium channel blocker and as such it can inactivate not only neuronal axons but also neuronal bodies. Consequently, it can not be excluded that besides its effects on the MCP, TTX could also inactivate nearby neurons potentially involved in CR expression. Although no known CR expression-related neurons are located in the vicinity of our MCP injection sites, to examine this possibility, one rabbit was injected with 3.5 nmol of muscimol at the site where TTX suppressed CRs. This treatment had no effect on CR incidence or latency.
The combined injections of GZ and TTX examined the involvement of the MCP in the expression of GZ-induced SLRs. All animals were injected first with GZ to induce SLRs and then with TTX to inactivate axons in the MCP. All drug injections in this experiment were administered at sites and with drug doses that were shown in prior experiments to induce SLRS (for GZ) and to abolish CRs (for TTX). As expected, GZ did not affect CR incidence but it did decrease eyeblink latency (Figs. 3A, 4E–F). Following TTX injections, mean CR incidence quickly decreased and remained suppressed through the end of the experiment (Fig. 4E, F9,27 = 6.08, p = 0.00012). In parallel to this effect, eyeblink latency significantly increased (Fig. 4F, F9,27 = 7.35, p = 0.000044). Control experiments consisted of GZ in the IN, but instead of TTX in the MCP, PBS was injected at the same time, depth, and volume as was TTX in each animal. In these experiments, GZ shortened CR latency as expected for the entirety of the experiment with no intervening effect of PBS on CR expression (Fig. 3B and and4E)4E) or eyeblink latency (Fig. 3B and and4F4F).
Our data demonstrated that injections of low doses of the GABAA antagonist GZ into the IN shorten the latencies of classically conditioned eyeblinks. Both SLRs and normal CRs were suppressed by inactivating the MCP. These findings suggest that normal activity in the MCP portion of the cerebellar mossy fiber system is essential for the expression of normal CRs and SLRs.
The intermediate cerebellum is involved in the acquisition and expression of conditioned eyeblinks. Current concepts suggest that the cerebellar cortex and deep cerebellar nuclei contribute to motor commands that generate CRs (Christian and Thompson, 2003; De Zeeuw and Yeo, 2005; Bracha et al., 2008). Since the intermediate cerebellar cortex and IN are serially connected and both receive CS and US information, the individual contribution of these two structures to CR generation have been difficult to establish. Garcia and Mauk (1998) proposed that the function of the IN could be uncovered by blocking the GABA-ergic input the IN receives from the cerebellar cortex. They reported that infusing the IN with the chloride channel blocker picrotoxin (PTX) evokes short-latency CRs, or SLRs. Garcia and Mauk hypothesized that SLRs are “unmasked” cerebellar nuclear CRs that are driven by direct projections from collaterals of mossy fibers to the IN. They further suggested that the function of the cerebellar cortex is to provide these nuclear CRs with an adaptive timing. To test this hypothesis, Ohyama et al. (2006) blocked the putative SLR-driving input from collaterals of mossy fibers by infusing the IN with glutamate antagonists. If SLRs were miss-timed nuclear CRs, and if they were driven solely by the direct mossy fiber nuclear input, one would expect that blocking this input would abolish all responses to the CS. Contrary to this prediction, Ohyama and colleagues found that glutamate antagonists did not eliminate CRs. The presence of CRs in animals with both Purkinje cell and mossy fiber inputs in the IN blocked was perplexing. It is possible that residual CRs in Ohyama et al.’s (2006) experiments were driven by extra-cerebellar circuits. However, it was also possible that their blocks of either mossy fibers or of Purkinje cell signals was incomplete and that residual signals in one of these inputs were sufficient to activate IN motor commands. Although the incomplete block of mossy fiber inputs in the IN can be excluded, it did not seem likely because two independent studies also failed to suppress CRs by blocking glutamate in the IN (Attwell et al., 2002; Aksenov et al., 2005). If SLRs are driven by incompletely blocked Purkinje cell inputs to the IN, one would expect that either a more extensive block of GABA neurotransmission in the IN or blocking mossy fiber inputs in the cerebellar cortex should suppress them. The feasibility of this scenario is supported by our previous investigations where we demonstrated that a more complete block of GABA-ergic neurotransmission in the IN with larger doses of PTX or GZ indeed abolishes CRs (Aksenov et al., 2004; Parker et al., 2009). Although this finding is consistent with the notion of SLRs being driven by the cerebellar cortex, it is inconclusive, because both PTX and GZ not only block signals from the cerebellar cortex, but they also disinhibit IN neurons to the point of saturation (Aksenov et al., 2004; Bracha et al., 2009). Clearly, elucidating cerebellar cortical involvement in SLR expression requires testing whether they are dependent on CS signals mediated through the cerebellar cortex.
CS signals are conveyed to the cerebellum from pontine nuclei via the middle cerebellar peduncle (Brodal and Jansen, 1946; Steinmetz et al., 1986; Steinmetz and Sengelaub, 1992; Hesslow et al., 1999). Consistent with this notion, we report here that inactivating the MCP with TTX severely suppressed normal CRs as well as SLRs, which are CRs facilitated by GZ administered to the IN. Before drawing conclusions from these observations, several alternate explanations of observed results should be considered. First of all, since TTX can inactivate neurons, did TTX inactivate eyeblink expression-related neurons that are not involved in cerebellar information processing? This seems unlikely not only because no known group of such neurons resides in the expected radius of TTX spread (Nilaweera et al., 2006), but also because the control injection of muscimol performed in one subject had no effect on CRs. Another possibility is that TTX inactivated not only the MCP, but also another fiber tract that is involved in CR expression. Indeed, injection sites in two rabbits were at places where spread of TTX to the superior cerebellar peduncle could not be excluded. However, injection sites in the other two animals were more rostral and ventral - at locations where drug diffusion to the brachium conjunctivum is unlikely. Another possibility is that TTX could have spread to the rubro-spinal tract which carries information from the red nucleus to eyeblink motoneurons. The rubro-spinal fibers, however, also appear to be located beyond the expected spread of TTX (Rosenfield et al., 1985).
Our findings suggest that SLRs are CRs that are facilitated by an increased tonic firing of IN neurons (Aksenov et al., 2004). They are not driven solely by mossy fiber projections to the IN because blocking this input with glutamate antagonists does not abolish CRs and does not abolish CS-related activity in IN neurons (Attwell et al., 2002; Aksenov et al., 2005; Ohyama et al., 2006). On the other hand, because SLRs are completely suppressed by MCP inactivation, they appear to be at least partly initiated by sensory signals and/or motor commands from incompletely blocked cerebellar cortical projections. Importantly, this conclusion assumes that inactivating the MCP does not significantly affect the tonic firing rate of IN neurons in a manner similar to the consequences of inactivating the inferior olivary cerebellar input (Zbarska et al., 2008). Our main conclusion predicts that inactivating the MCP should suppress CS and CR-related neuronal activity in the GZ pre-treated IN and that MCP inactivation does not significantly affect the tonic activity of IN neurons. These important issues will be examined in future studies.
Experiments were performed on 4 male New Zealand White Rabbits (Harlan; Indianapolis, IN) weighing 2.5–3.0 kg (3–4 months old at time of surgery). Rabbits were housed individually on a 12-hour light/dark cycle and provided food and water ad libitum. All experiments were performed in accordance with the National Institutes of Health’s “Principles of Laboratory Animal Care” (publication No. 86-23, revised 1985), the American Physiological Society’s “Guiding Principles in the Care and Use of Animals,” and the protocol approved by Iowa State University’s Committee on Animal Care.
Using aseptic techniques, surgery was performed on naive rabbits anesthetized with a mixture of ketamine (50 mg/kg), xylazine (6 mg/kg) and acepromazine (1.5 mg/kg). The head was secured in a stereotaxic apparatus with lambda positioned 1.5 mm ventral to bregma. Two stainless steel guiding tubes (28 gauge thin-wall, 1 mm apart) were stereotaxically implanted 0.5 mm dorsal to the expected location of the anterior IN ((0.69x + 4.8) - x mm rostral from lambda, x being the horizontal distance between bregma and lambda in mm; 5.3 mm lateral and 13.5 mm ventral to lambda). Targeting the MCP was accomplished using the following coordinates measured from bregma: 12.5 mm caudal, 4.0 mm lateral, and 17 mm ventral. To protect the patency of guide tubes, a 33 gauge stainless steel stylet was inserted into each guiding tube in-between experiments. The guide tube, skull anchor screws, and a small Delrin block designed to accommodate an airpuff delivery nozzle and eyeblink sensor were secured in place with dental acrylic. All animals were treated with antibiotics for 5 days during the recovery period following surgery.
Following recovery from surgery, rabbits were adapted to a restraint box for three 30 minutes/day sessions. Box-adapted rabbits were trained in the standard classical conditioning paradigm until they reached at least 90 % CRs for 3 consecutive days. The conditioned stimulus (CS) was an 85-db, 450-ms, 1-Hz tone, superimposed on a continuous 70-dB white noise background. The CS co-terminated with a 40-psi, 100-ms airpuff unconditioned stimulus (US) directed to the left eye. The inter-stimulus interval was 350 ms and each training session consisted of 100 trials presented in pseudorandom, 15–25 sec inter-trial intervals. All experiments were conducted in a sound-attenuated chamber.
Injections were delivered via a 33-gauge stainless steel injection needle which was connected via transparent Tygon tubing to a 10-μL Hamilton syringe. The injection needle was inserted in the guide tube prior to beginning each experiment. A pre-injection period of 40 trials was presented to assess baseline eyeblink performance and to detect any insertion effect. Following the pre-injection period, drug micro-injections were manually administered at a rate of 0.5 μL/min. To assess the drug effect, CS + US training continued for an additional 160 trials.
The present study consisted of three parts. The animals were first injected in the interposed nuclei (IN) to determine the location and the dosage of the GABAA antagonist gabazine (GZ) that would shorten the onset latency of CRs, i.e., produce SLRs (Parker et al., 2009). GZ was injected at locations in the IN where muscimol (1.75 nmol) completely suppressed conditioned eyeblinks (Bracha et al., 1994). Once the optimal GZ dosage and location were found in the IN, the MCP was injected the next experiment day with the sodium channel blocker TTX to determine a site at which MCP inactivation abolished CRs. In the third part, these two experiments were combined to test the effect of blocking the MCP on SLR expression. First, GZ was injected at locations in the IN where muscimol (1.75 nmol) completely suppressed conditioned eyeblinks (Bracha et al., 1994) and then TTX was injected where 3 μL of 4% Lidocaine had the same effect in the MCP. Immediately prior to each injection experiment, GZ was dissolved in artificial cerebral spinal fluid (aCSF), TTX was dissolved in phosphate-buffered saline (PBS), and the pH of both drugs was adjusted to 7.4 ± 0.1. In control experiments, an equal volume of drug vehicle was injected using the same protocol.
Each rabbit’s behavior was monitored using an infrared video system installed in the experiment chamber. Eyelid movements were recorded using a frequency-modulated infrared sensor that measures infrared light reflected from the eye and peri-orbital region (Ryan et al., 2006). The sensor was attached to the head implant before every experiment. The output of the sensor was amplified, digitized (25 kHz, 12-bit A/D converter), and stored in a PC-based data acquisition system. During each trial, 1400 ms of the signal were recorded, beginning 250 ms before the onset of the CS and extending for 800 ms beyond the US onset.
Eyeblink responses from each trial were examined off-line for the presence of CRs within the time window between CS and US onsets. The threshold for eyeblink detection was set to 5 standard deviations of the baseline signal noise, which in the present setup corresponded to approximately a 0.15 mm decrease in the eyelid aperture. Means of eyeblink latency and CR incidence were calculated for consecutive blocks of 20 trials. The pooled data from individual rabbits were statistically analyzed using repeated measures ANOVA. Reported F-ratios and their p-values refer to the differences between drug and vehicle in the pre-injection period versus the overall drug effect post injection and in some cases versus a given post-injection block. All group data were reported as mean ± standard error of mean, and an alpha level = 0.05 was used to declare significance. All statistical analyses were performed using Statsoft Statistica software.
Upon the conclusion of experimentation, rabbits were deeply anesthetized with a concentrated cocktail of ketamine (100 mg/kg), xylazine (12 mg/kg), and acepromazine (3 mg/kg). The injection sites were marked by injecting 1 μL of tissue-marking dye. Animals were perfused transcardially with 1 L of PBS followed by 1 L of tissue fixative (10 % neutral buffered formalin, NBF). Carefully excised brains were stored in a solution of 30 % sucrose and 10 % NBF and subsequently sectioned coronally at 50 μm on a freezing microtome. The sections were mounted onto gelatin-coated slides, and once dry, stained with luxol blue and neutral red. Using bright light microscopy, injection locations were determined and plotted on standard sections of the rabbit cerebellum.
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