Stereological Analysis of Prefrontal GABAergic Interneurons following Perinatal Hippocampal Lesion in Rhesus monkeys Open Access

Libecap, Timothy Justin (2016)

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GABA is the primary inhibitory neurotransmitter in the mammalian brain. Under normal working circumstances, GABA plays a fundamental role in the dorsolateral prefrontal cortex (dlPFC). In particular, the parvalbumin class of GABA interneurons is involved in sustaining the normal neuron signaling that maintains proper functionality. The dlPFC is a brain region responsible for executive functioning and undergoes a protracted development. Disruption to its development may play a role in working memory impairment and other cognitive deficits associated with neurodevelopmental disorders including schizophrenia. Further, the hippocampus is a region in the medial temporal lobe that develops early in life and has been implicated in the development of the dlPFC as a result of their synaptic connections. In our study, we used tissue harvested from four control and four experimental adult rhesus monkeys that sustained bilateral neonatal hippocampal lesions of varying degree in order to better understand the role of the hippocampus in the development of the dlPFC. Specifically, we analyzed the relative density of parvalbumin-immunoreactive GABAergic interneurons in Brodmann Area 46d of lesioned (Neo-H) and non-lesioned monkeys (Neo-C). We used immunohistochemistry, Giemsa counterstain, and principles of stereology to visualize and analyze the density of PV-labeled neurons. We found a significantly higher density of parvalbumin-positive neurons within the dlPFC of the Neo-H monkeys relative to the Neo-C monkeys, both between the left hemispheres of the two groups and across the groups when both hemispheres were considered together. These results were confirmed by estimations of total PV-positive neuron populations in area 46d. Moreover, a correlation analysis between the hippocampal volume reduction and PV-positive cell density demonstrated a strong positive correlation such that a more greatly reduced hippocampus corresponded with a greater density of PV-stained cells. Our results are consistent with functional connectivity and behavioral data taken from the same monkeys. These findings have implications for how early life lesions of the hippocampus may affect vulnerable structures and disrupt cognitive processing. This work suggests that structural analysis of lesion studies supplements behavioral data to support a more comprehensive understanding of complex circuitry underlying psychiatric disease etiology.

Table of Contents

Introduction. 1

GABA and GABAergic neurons. 1

GABA alterations in schizophrenia. 2

Regions of interest in the current study. 6

Hippocampal changes in schizophrenia. 7

Rhesus monkey model. 8

Predictions. 9

Significance. 9

Methods. 10

Subjects and lesions. 10

Brain harvesting. 11

Designation of layers. 12

Data analysis. 13

Randomization of tissue sections. 13

Cell count. 14

Statistical analysis. 15

Effect size. 16

Layer area and Volume. 16

Coefficient of Error. 17

Results. 18

Parvalbumin Analysis. 18

Parvalbumin-immunoreactive neuron density. 18

PV density effect size. 19

Total parvalbumin cell estimate. 20

Correlation between hippocampal lesion size and average PV cell density. 21

Coefficient of error. 22

Discussion. 22

Overview. 22

Possible mechanisms of GABA circuit alteration. 23

Implications of parvalbumin circuitry alteration. 25

Lateralized effects of hippocampal lesion. 27

Limitations. 29

Functional significance. 30

Conclusions. 32

Future directions. 32

References. 35

Figures. 45

1: Macroscopic view of hippocampus and prefrontal cortex. 46

2. Dorsal lateral prefrontal cortex. 46

3. Photo reticle. 47

4. Optical fractionator. 47

5. Stereological setup. 48

6. Cortical layers. 49

7. Antigen retrieval. 49

8. Immunohistochemistry. 50

9. GABAergic circuitry. 51

10. Differential shrinkage. 51

11. GABAergic developmental trajectory. 52

12. Tissue series. 53

13. Parvalbumin-stained cells. 54

14. Somatostatin-stained cells. 54

15. PV-cell density by hemisphere. 56

16. PV-cell density by group. 56

17. Total PV-positive cell count by hemisphere. 57

18. Total PV-positive cell count by group. 58

19. Lesion percentage by PV count average. 59

20. Summary statistics. 60

21. Hemispheric volumes. 60

22. Coefficient of error. 60

23. Antigen retrieval manipulations. 62

24. PV-positive cell density calculations. 63

25. PV-positive cell estimation calculations. 63

26. Lesion percentage. 64

27. Volume reduction. 64

28. Monkey behavioral data. 65

Appendix. 62

CE equations. 66

Supplemental writings. 68

Other GABAergic Alterations in Schizophrenia. 69

GABA. 71

Histological Processing: GRα2 and Somatostatin. 72

Somatostatin Staining. 75

GABA Receptor Alpha Two Subunit Staining. 75

Coefficient of Error. 75

Stereology. 76

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