Literature context: 7279, Santa Cruz Biotechnology; RRID:AB_2110230) detected in the same distribut
The basal forebrain (BF) receives afferents from brainstem ascending pathways, which has been implicated first by Moruzzi and Magoun (1949) to induce forebrain activation and cortical arousal/waking behavior; however, it is very little known about how brainstem inhibitory inputs affect cholinergic functions. In the current study, glycine, a major inhibitory neurotransmitter of brainstem neurons, and gliotransmitter of local glial cells, was tested for potential interaction with BF cholinergic (BFC) neurons in male mice. In the BF, glycine receptor α subunit-immunoreactive (IR) sites were localized in choline acetyltransferase (ChAT)-IR neurons. The effect of glycine on BFC neurons was demonstrated by bicuculline-resistant, strychnine-sensitive spontaneous IPSCs (sIPSCs; 0.81 ± 0.25 × 10-1 Hz) recorded in whole-cell conditions. Potential neuronal as well as glial sources of glycine were indicated in the extracellular space of cholinergic neurons by glycine transporter type 1 (GLYT1)- and GLYT2-IR processes found in apposition to ChAT-IR cells. Ultrastructural analyses identified synapses of GLYT2-positive axon terminals on ChAT-IR neurons, as well as GLYT1-positive astroglial processes, which were localized in the vicinity of synapses of ChAT-IR neurons. The brainstem raphe magnus was determined to be a major source of glycinergic axons traced retrogradely from the BF. Our results indicate a direct effect of glycine on BFC neurons. Furthermore, the presence of high levels of plasma membrane glycine transporters in the vicinity of cholinergic neurons suggests a tight control of extracellular glycine in the BF.SIGNIFICANCE STATEMENT Basal forebrain cholinergic (BFC) neurons receive various activating inputs from specific brainstem areas and channel this information to the cortex via multiple projections. So far, very little is known about inhibitory brainstem afferents to the BF. The current study established glycine as a major regulator of BFC neurons by (1) identifying glycinergic neurons in the brainstem projecting to the BF, (2) showing glycine receptor α subunit-immunoreactive (IR) sites in choline acetyltransferase (ChAT)-IR neurons, (3) demonstrating glycine transporter type 2 (GLYT2)-positive axon terminals synapsing on ChAT-IR neurons, and (4) localizing GLYT1-positive astroglial processes in the vicinity of synapses of ChAT-IR neurons. The effect of glycine on BFC neurons was demonstrated by bicuculline-resistant, strychnine-sensitive spontaneous IPSCs recorded in whole-cell conditions.
Literature context: SC-17279 RRID:AB_2110230 GlyRÎ±3 Rai
Amacrine cells comprise ∼ 30 morphological types in the mammalian retina. The synaptic connectivity and function of a few γ-aminobutyric acid (GABA)ergic wide-field amacrine cells have recently been studied; however, with the exception of the rod pathway-specific AII amacrine cell, the connectivity of glycinergic small-field amacrine cells has not been investigated in the mouse retina. Here, we studied the morphology and connectivity pattern of the small-field A8 amacrine cell. A8 cells in mouse retina are bistratified with lobular processes in the ON sublamina and arboreal dendrites in the OFF sublamina of the inner plexiform layer. The distinct bistratified morphology was first visible at postnatal day 8, reaching the adult shape at P13, around eye opening. The connectivity of A8 cells to bipolar cells and ganglion cells was studied by double and triple immunolabeling experiments by using various cell markers combined with synaptic markers. Our data suggest that A8 amacrine cells receive glutamatergic input from both OFF and ON cone bipolar cells. Furthermore, A8 cells are coupled to ON cone bipolar cells by gap junctions, and provide inhibitory input via glycine receptor (GlyR) subunit α1 to OFF cone bipolar cells and to ON A-type ganglion cells. Measurements of spontaneous glycinergic postsynaptic currents and GlyR immunolabeling revealed that A8 cells express GlyRs containing the α2 subunit. The results show that the bistratified A8 cell makes very similar synaptic contacts with cone bipolar cells as the rod pathway-specific AII amacrine cell. However, unlike AII cells, A8 amacrine cells provide glycinergic input to ON A-type ganglion cells.
Inhibitory glycine receptors (GlyRs) are composed of homologous α- (α1-4) and β-subunits. The β-subunits (GlyRβ) interact via their large cytosolic loops with the postsynaptic scaffolding protein gephyrin and are therefore considered essential for synaptic localization. In situ hybridization studies indicate a widespread distribution of GlyRβ transcripts throughout the mammalian central nervous system (CNS), whereas GlyRα mRNAs and proteins display more restricted expression patterns. Here we report the generation of a monoclonal antibody that specifically recognizes rodent GlyRβ (mAb-GlyRβ) and does not exhibit crossreactivity with any of the GlyRα1-4 subunits. Immunostaining with this antibody revealed high densities of punctate GlyRβ immunoreactivity at inhibitory synapses in mouse spinal cord, brainstem, midbrain, and olfactory bulb but not in the neocortex, cerebellum, or hippocampus. This contrasts the abundance of GlyRβ transcripts in all major regions of the rodent brain and suggests that GlyRβ protein levels are regulated posttranscriptionally. When mAb-GlyRβ was used in double-labeling experiments with GlyRα1-, α2-, α3-, or α4-specific antibodies to examine the colocalization of GlyRβ with these GlyR subunits in the mouse retina, >90% of the GlyRα1-3 clusters detected were found to be GlyRβ-immunoreactive. A subset (about 50%) of the GlyRα4 puncta in the inner plexiform layer, however, was found to lack GlyRβ and gephyrin immunostaining. These GlyRα4-only clusters were apposed to bassoon immunoreactivity and hence synaptically localized. Their existence points to a gephyrin-independent synaptic localization mechanism for a minor subset of GlyRs.
Retinas of macaque monkeys were immunostained for glycogen phosphorylase (glypho). Glypho was localized to regular and displaced amacrine cells. Their processes occupied two narrow strata within the inner plexiform layer (IPL). The labeling pattern is reminiscent of cholinergic amacrine cells; however, double immunostaining of the retinas for choline acetyltransferase and glypho revealed two different cell populations. Intracellular injection of DiI showed that glypho-immunoreactive amacrine cells are wide-field amacrine cells with straight, radially oriented, and sparsely branched dendrites. The density of the cells increased from approximately 70/mm(2) in the peripheral retina to approximately 700/mm(2) in the central retina. The regular glypho-immunoreactive amacrine cells branch in sublamina 2 of the IPL, where they receive input from OFF-cone bipolar cells. The displaced cells branch in sublamina 3/4 and receive input from ON-cone bipolar cells. This suggests that the regular cells are OFF-cells and the displaced cells are ON-cells. The cells express gamma-aminobutyric acid (GABA)-like immunoreactivity and receive glycinergic input through synapses expressing preferentially the glycine receptor alpha2 subunit. The close proximity of the dendritic strata of glypho-immunoreactive amacrine cells, cholinergic amacrine cells, and direction-selective ganglion cells suggests a possible role of the cells in the generation of direction-selective light responses of the monkey retina.