Korean J Physiol Pharmacol.  2012 Feb;16(1):17-24. 10.4196/kjpp.2012.16.1.17.

The Characteristics of Supramammillary Cells Projecting to the Hippocampus in Stress Response in the Rat

• Choi WK
• Wirtshafter D
• Park HJ
• Lee MS
• Her S
• Shim I

• KMID: 2285435
• DOI: http://doi.org/10.4196/kjpp.2012.16.1.17

Abstract

The hypothalamus-pituitary-adrenocortex (HPA) axis is the central mediator of the stress response. The supramammillary (SuM) region is relatively unique among the hypothalamic structures in that it sends a large, direct projection to the hippocampal formation. It has been shown that mild stress could activate the SuM cells that project to the hippocampus. However, the role of these cell populations in modulating the stress response is not known. The present study examined the effect of stress on different populations of SuM cells that project to the hippocampus by injecting the fluorescent retrograde tracer, fluorogold (FG), into the hippocampus and utilizing the immunohistochemistry of choline acetyltransferase (ChAT), corticotrophin releasing factor (CRF), serotonin (5-HT), glutamate decarboxylase (GAD), tyrosine hydroxylase (TH) and NADPH-d reactivity. Immobilization (IMO) stress (2 hr) produced an increase in the expression of ChAT-immunoreactivity, and tended to increase in CRF, 5-HT, GAD, TH-immunoreactivity and nitric oxide (NO)-reactivity in the SuM cells. Fifty-three percent of 5-HT, 31% of ChAT and 56% of CRF cells were double stained with retrograde cells from the hippocampus. By contrast, a few retrogradely labeled cells projecting to the hippocampus were immunoreactive for dopamine, gamma-aminobutyric acid (GABA) and NO. These results suggest that the SuM region contains distinct cell populations that differentially respond to stress. In addition, the findings suggest that serotonergic, cholinergic and corticotropin releasing cells projecting to the hippocampus within the SuM nucleus may play an important role in modulating stress-related behaviors.

Keyword

Immobilization stress; Supramammillary region; Hippocampus; Fluorogold; Immunohistochemistry

MeSH Terms

Adrenocorticotropic Hormone
Animals
Axis, Cervical Vertebra
Choline O-Acetyltransferase
Dopamine
gamma-Aminobutyric Acid
Glutamate Decarboxylase
Hippocampus
Immobilization
Immunohistochemistry
Nitric Oxide
Rats
Serotonin
Tyrosine 3-Monooxygenase
Adrenocorticotropic Hormone
Choline O-Acetyltransferase
Dopamine
Glutamate Decarboxylase
Nitric Oxide
Serotonin
Tyrosine 3-Monooxygenase
gamma-Aminobutyric Acid

Figure

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  Fig. 1 Camera lucida drawing of injection site of the hippocampus (A). Marked spot is enlarged in (B); (100×).

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  Fig. 2 Schematic drawings of the region of the supramammillary complex (A) and low magnification photographs showing the distribution of the retrogradely labeled cells following injections of flourogold into the hippocampus (B). MT, mammillothalamic tract; PH, posterior nucleus; SMT, submammillothalmic nucleus; SuMM, supramammillary medial; SuML, supramammillary lateral; MnM, medial mammillary median; MM, medial mammillary nucleus, medial part; ML, medial mammillary nucleus, lateral part; LM, lateral mammillary nucleus; PH, posterior nucleus. Scale bar; 500µm.

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  Fig. 3 Low (A~C) and high magnification (D~F) microphotographs showing the distribution of choline acetyltrasferase (ChAT)-like immunoreactive cells (A, D; in green) and retrogradely labeled cells (B, E; in red) after acute IMO stress. The overlay of images A+B, D+E is shown in C, F, respectively where yellow color illustrates co-localization of ChAT with retrogradely labeling. Scale bar in A, 100µm; D, 20µm. MT, mammillothalamic tract.

• 

  Fig. 4 Low (A~C) and high magnification (D~F) microphotographs showing the distribution of corticotropin releasing factor (CRF)-like immunoreactive cells (A, D; in green) and retrogradely labeled cells (B, E; in red) after acute IMO stress. The overlay of images A+B, D+E is shown in C, F, respectively where yellow color illustrates co-localization of CRF with retrogradely labeling. Scale bar in A, 100µm; D, 20µm. MT, mammillothalamic tract.

• 

  Fig. 5 Low (A~C) and high magnification (D~F) microphotographs showing the distribution of serotonin (5-HT)-like immunoreactive cells (A, D; in green) and retrogradely labeled cells (B, E; in red) after acute IMO stress. The overlay of images A+B, D+E is shown in C, F, respectively where yellow color illustrates colocalization of 5-HT with retrogradely labeling. Scale bar in A, 100µm; D, 20µm. MT, mammillothalamic tract.

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  Fig. 6 Microphotographs showing the distribution of glutamate decarboxylase (GAD67) (A; in green) and tyrosine hydroxylase (TH) (D; in green) with retrogradely labeled cells (FG) (B, E; in red) after acute IMO stress. The overlay of images A+B and D+E is shown in C and F, respectively, where a few GAD67 and TH-like immuoreactive neurons were colocalized with retrogradely labeling (arrow). Scale bar in A and D; 100µm. MT, mammillothalamic tract.

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  Fig. 7 Microphotographs showing NADPH-d (A), and retrogradely labeled cells (B) in the supramammillary region after acute IMO stress. Scale bar in A; 50µm, MT, mammillothalamic tract.

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  Fig. 8 Summary diagram depicting cell number of ChAT, 5-HT and CRF immunoreactive- and double-labeled cells in the SuM region after acute IMO stress. DL, double-labeled cells. **p>0.001 versus the Normal group.

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