A novel GABAergic afferent input to the pontine reticular formation: the mesopontine GABAergic column

Abstract

Pharmacological manipulations of gamma-aminobutyric acid (GABA) neurotransmission in the nucleus pontis oralis (PnO) of the rat brainstem produce alterations in sleep/wake behavior. Local applications of GABA(A) receptor antagonists and agonists increase REM sleep and wake, respectively. These findings support a role for GABAergic mechanisms of the PnO in the control of arousal state. We have been investigating sources of GABA innervation of the PnO that may interact with local GABA(A) receptors in the control of state. Utilizing a retrograde tracer, cholera toxin-B subunit (CTb), injected into the PnO and dual-label immunohistochemistry with an antibody against glutamic acid decarboxalase-67 (GAD67), we report on a previously unidentified GABAergic neuronal population projecting to the contralateral PnO appearing as a column of cells, with long-axis in the sagittal plane, extending through the midbrain and pons. We refer to these neurons as the mesopontine GABAergic column (MPGC). The contiguous, columnar, anatomical distribution suggests operation as a functional neural system, which may influence expression of REM sleep, wake and other behaviors subserved by the PnO.

Fig. 1

Representative example of a CTb injection into the caudal aspects of the PnO. The injection site is visualized by the large, dark spot (arrow) immuno-labeled for CTb in this coronal section counter-stained with neutral red. Bar represents 1 mm.

Fig. 2

(A-C) Examples of immuno-labeled neurons in the MPGC. (A) Bright-field immunoperoxidase labeled neurons in the PnO on the side contralateral to the CTb injection. CTb is labeled with nickel-intensified DAB (bluish-black) and GAD67 with DAB (yellowish-brown). Double-labeled, CTb/GAD67 somata are indicated by large arrows. Single-labeled, CTb-only somata are indicated by small arrows. Several GAD67-only somata appear and are not indicated by arrows. (B) Laser scanning fluorescent image of a double-labeled neuron in the midbrain portion of the column. Panels, B1 and B2, are the individual color channels; CTb, green and GAD67, red. Panel B3 is the merged image, colocalization is yellow. (C) Laser scanning fluorescent z-stack of a field in the PnO using a 1 μm focal plane. The four panels, C1-4, are a series of every second image in the stack. Colors are as they appear in (B). Small arrows in C3 indicate double-labeled, CTb/GAD67 somata and large arrow, CTb-only. (A-C) Bar indicates scale.

Fig. 3

Schematic diagram of the distribution of double-labeled, CTb/GAD67 somata (filled circles) and single-labeled, CTb-only somata (open circles). (A-H) Coronal sections, anterior to posterior, of a representative animal through the region of the MPGC on the side contralateral to the CTb injection. Sections are labeled with their approximate distance (mm) posterior to the Bregma suture. Each symbol represents the count of three somata on that section. Diagrams adapted from Paxinos and Watson (1997).