Functional Dissociation Along The Rostrocaudal Axis Of Japanese Quail Hippocampus Part 2

Histology

Following testing, subjects were transported to a procedure room, anesthetized with isoflurane, decapitated, and brains were extracted and flash frozen in 2-methylbutane (Sigma Aldrich, Oakville, ON). Coronal sections were cut at a thickness of 30 μm using a CM3050 cryostat (Leica) and thaw-mounted onto Superfrost Plus™ slides (Thermo Scientific, Waltham, MA). 

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Every 6th section was then stained using Methyl Green to observe the placement and extent of the lesions under a light microscope. Thaw-mounted sections were fixed for 5 min in buffered 4% formaldehyde, rinsed in double-distilled water for 5 min, then incubated in Methyl Green for 5 minutes at room temperature. Slides were then dehydrated in a graded series of ethanol, cleared with Neo-clear, and embedded in Neo-mount (all histology reagents were obtained from Sigma Aldrich, Oakville, ON).

Analysis

Two quail died during surgery, while another 2 were excluded for lack of movement in at least one of the 2 tests, yielding final data on 25 quail (9 rHp, 7 cHp, 7 sham).

Analysis of the CFC data was conducted using a repeated-measures analysis of variance (ANOVA) of the time spent freezing during the first 2 minutes of each trial using context (i.e., acoustically-paired vs. control) and time (i.e., immediate vs. remote) as within-subject factors and group (i.e., rHp, cHp, and sham) as between-subject factors. As an additional control, the time spent freezing in each context before any acoustical stimulation was also compared using a 2 (context) x 3 (group) ANOVA.

In the YMD, the time spent within each arm was quantified as a proportion of total exploration time. The subject was considered to be exploring an arm if their entire torso was inside the arm. The time spent exploring the novel (TN) and familiar (TF) arms (excluding the start arm) was converted into a discrimination ratio (DR) for each subject, as follows: DR = (TNTF)/(TN+TF). These DRs were compared across groups using a one-way ANOVA.

Post hoc tests were conducted using Tukey’s HSD. All statistical tests were conducted using JASP [38].

Results

Lesions to either the rostral or caudal hippocampus spare contextual fear conditioning in quail

Analysis of CFC (Fig 2) showed no significant difference between contexts (F1,20 = 1.03; p = 0.32) or groups (F2,20 = 1.42; p = 0.27) before acoustic stimulation, showing that the surgeries did not induce any baseline differences in freezing behavior. Examining the time spent freezing during the trials following stimulation failed to show a significant main effect of time (F1,20 = 1.03; p = 0.32) or of the group (F2,20 = 0.33; p = 0.73). However, a significant effect of context (F1,20 = 7.83; p = 0.01) as well as a significant time-by-context interaction (F1,20 = 9.35; p < 0.01) were observed. 

This pattern of results suggests that quail across all groups in the immediate test selectively froze in the context paired with the acoustic stimulus and not in the control environment (paired vs. unpaired context: p < 0.05 for all groups), indicating that they can discriminate between the two contexts and retain a memory for the context in which the acoustic stimulus had been presented. 

Thus, it seems that the spared Hp in either lesion group supports this behavior. In contrast, 24 hours later, freezing had diminished to the point at which no significant difference could be observed in any group (p > 0.05 for all groups), suggesting that the memory had degraded for all quail.

Quail rostral hippocampus is critical for Y-maze discrimination

Analysis of the YMD (Fig 3), yielded a significant effect of condition (F2,20 = 3.99; p = 0.03). Post-hoc tests showed that while rHp-lesioned quail performed significantly worse than shams (p = 0.04), cHp-lesioned quail did not (p = 0.12). This pattern of results suggests that, like mammals, the rHp of quail may disproportionately support spatial learning tasks.

Discussion

The current results are the first to report functional segregation along the rostrocaudal axis of the avian Hp. These results partially confirm the existence of a gradient along the rostrocaudal axis that is in some ways comparable to the mammalian dorsoventral axis. In particular, we observe that the rHp is necessary for the identification of spatial novelty during YMD. This observation is consistent with results produced in rodents completing comparable tasks following lesions to the dHp ([10, 39]; but see [40]). 

Moreover, the current results are consistent with reports of a gradient of spatial information content in avian Hp, with the greatest spatial information represented in principal cells of the rHp [29]. This pattern, which mirrors the change in information content observed along the rodent dorsoventral axis [41] further bolsters the body of evidence demonstrating that the most rostral extent of the Hp disproportionately supports high-resolution spatial information processing across both Aves and Mammalia.

The current data replicate previous observations [37] that, like rodents, quail will react to novel stimuli including novel spatial locations by approaching the novel stimulus, even when novelty is relative (i.e., the novel stimulus has been seen but fewer times or longer ago). It is worth noting, however, that during the test trial of YMD, the novel arm has not been seen for 24 hours. At this delay, quail also does not selectively freeze to a previously fear-associated context, suggesting that 24 hours is beyond the spatial memory capacity of quail.

The observation of selective freezing at shorter intervals during CFC training shows that, like mammals, quail can associate a noxious stimulus with the context in which it was presented, leading to quail selectively freezing in the stimulus-associated context. The observation that this association is preserved following either rHp or cHp lesions is inconsistent with the majority of data on the mammalian vHp. Several conclusions are possible given this observation. The caudal lesions may make here spare enough tissue to mediate CFC. 

Some data suggest that the functional equivalent of the vHp may be considerably larger in a bird than in a mammal. Anatomical studies in pigeons [26] report that input from the nucleus taeniae of the amygdala is absent in the rostral third of the HF, but widespread in the caudal two-thirds of this region. These widespread connections suggest that the majority of the avian Hp is homologous to the ventral mammalian Hp, and the cHp lesions conducted here were not sufficient to remove this distributed structure in its entirety. This may be particularly true for Japanese quail (or Galliformes in general) relative to other avian orders. This suggestion is consistent with observations of species differences in spatial information processing. Bird species vary considerably as to the extent of the high-resolution spatially-tuned dHp analog [29]. 

It would be intuitive, then, to hypothesize that birds with a smaller rHp (e.g., birds that do not cache food) would have a proportionately larger cHp. Recent physiological data showing a lack of place cells in quail even in relatively rostral coordinates further suggests that the rHp may be exceptionally small in quail, even among non-food-caching birds. By extension, CHP may be exceptionally large in these birds. This proposal is consistent with a recent MRI-based quail atlas that supports the presence of a large cHp [42]. Functionally, the idea that quail may have an exceptionally large “emotional processing” center is also consistent with the use of quail as a model of stress reactivity [43, 44]. Finally, it remains possible that CFC is entirely Hp-independent in birds. There is data in rodents implicating the prefrontal cortex in mediating CFC (reviewed by [45]). 

The NCL, the equivalent structure in birds, also plays a role in processing context [46, 47]. It is possible that in birds, unlike mammals, the NCL is capable of mediating CFC even in the face of extensive damage to the hippocampus. This possibility seems unlikely, however, given the extensive homology found between the avian and mammalian hippocampus to date. This is made even more unlikely by two additional observations. The first is that, even in mammals, the extent to which the dHp or vHp are selectively required for CFC is affected by relatively subtle changes in protocol [48]. Notably, the presence of a discrete cue that signals the presence of shock during training seems to make the subsequent freezing when exposed to only the context depend more on the vHp. This cue was absent in the current experiment. 

Additionally, at least one experiment has reported preserved CFC in rodents with selective lesions to either the dHp or vHp [12]. Importantly, in this study removal of the entire Hp dramatically reduced freezing in a shock-associated context, even though selective lesions to either half of the Hp left CFC intact. Collectively, these observations make it far more likely that, like mammals under some conditions, CFC in quail can be supported by either the rHp or cHp alone.

Despite remaining open questions concerning the extent and functional heterogeneity of the CHP, the current results demonstrate that, like its mammalian homolog, the avian Hp is functionally heterogeneous, with its rostral portion specialized for computations that support spatial learning and memory.

Author Contributions

Conceptualization: Chelsey C. Damphousse, Noam Miller, Diano F. Marrone.

Data curation: Chelsey C. Damphousse, Diano F. Marrone.

Formal analysis: Chelsey C. Damphousse, Diano F. Marrone.

Funding acquisition: Noam Miller, Diano F. Marrone.

Investigation: Chelsey C. Damphousse, Diano F. Marrone.

Methodology: Chelsey C. Damphousse, Noam Miller, Diano F. Marrone.

Project administration: Chelsey C. Damphousse, Diano F. Marrone.

Supervision: Noam Miller, Diano F. Marrone.

Writing – original draft: Chelsey C. Damphousse.

Writing – review & editing: Chelsey C. Damphousse, Noam Miller, Diano F. Marrone.

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