Co-existence of GABA/glutamate and acetylcholine in pedunculopontine tegmental inputs to caudal intralaminar nuclei but not sensory relay nuclei in monkey thalamus
Yedidya Saiman, Jean Francois Pare, Mamadou Sidibe, and Yoland Smith
Department of Biology, Yeshiva University, New York, NY
Division of Neuroscience, Yerkes Regional Primate Research Center, Atlanta, GA
Department of Neurology, Emory University, Atlanta, GA

Abstract

The thalamus receives massive cholinergic inputs from the pedunculopontine tegmental nucleus (PPN). To further characterize the chemical phenotype of PPN cholinergic inputs to the thalamus we performed a detailed electron microscopic analysis of GABA and glutamate localization in cholinergic afferents to caudal intralaminar nuclei and sensory relay nuclei in monkey. This was achieved using post-embedding immunogold for GABA or glutamate combined with immunoperoxidase labeling for choline acetyltransferase (ChAT). These experiments revealed a striking degree of chemical heterogeneity between cholinergic inputs to intralaminar versus relay nuclei. In the centromedian/parafascicular complex (CM/PF) 40-60% of ChAT-containing terminals displayed GABA immunoreactivity whereas the cholinergic terminals in the lateral geniculate (LGen) and ventroposterolateral (VPL) nuclei were devoid of GABA labeling. On the other hand preliminary data indicates that 20-80% of cholinergic terminals in both intralaminar and relay nuclei are enriched in glutamate. These findings emphasize the differential chemical phenotype of ascending brainstem cholinergic inputs to intralaminar versus sensory relay nuclei in the primate thalamus which paves the way for detailed functional studies of complex cholinergic/GABAergic and cholinergic/glutamatergic modulation of thalamic activity during changes in states of arousal.

Introduction

The thalamus is a key structure for transmitting specific information to the cerebral cortex. Through the modulation of cortical activity the thalamus also plays a significant role in regulating sleep-wake cycles. The upper brainstem and particularly the pedunculopontine nucleus/lateral tegmental (LDT) region provides a massive cholinergic input to the thalamus. Of the ~20 000 cholinergic neurons stemming from the PPN close to 50% are directed to the thalamus. The thalamus is comprised of two categories of nuclei. Relay nuclei such as the lateral geniculate (LGen) and ventroposterolateral (VPL) nuclei are essential for brain function with each nucleus playing a distinct role in perception volition or cognition. These nuclei provide input to restricted and specific areas of the cerebral cortex; therefore each sense is controlled by a specific thalamic nucleus. On the other hand diffuse projecting nuclei affect wide regions of the cerebral cortex and are essential in arousal and regulating cortical excitability. They receive inputs from many sources consequently projecting widely within the cerebral cortex. These nuclei are part of the intralaminar nuclear group and include the centromedian (CM) parafascicular (PF) and parafascicular dorsal lateral (Pf-dl) thalamic nuclei. Previous studies have shown that retrogradely labeled neurons are found in the PPN when tracers are injected into the thalamus. While the majority of large neurons coming from the PPN are cholinergic significant proportions are indeed non-cholinergic. Additional neurons displaying both ChAT and glutamate immunoreactivity have been identified within the PPN and surrounding nuclei.

Methods and Materials

Animals: adult rhesus monkeys

Histology

The monkeys were perfused with 4% paraformaldehyde and 0.05% glutaraldehyde. Sections were cut and prepared for electron microscopy (EM).

Pre-embedding Immunocytochemistry

A primary antibody rabbit anti-ChAT (Chemicon) was used at a concentration of 1:15 000.A secondary antibody goat anti-rabbit coupled to a biotin molecule (Vector) was used at a concentration of 1:200. The antibodies were revealed using the avidin biotin complex (ABC) method and diaminobenzidine (DAB).

Post-embedding Immunocytochemistry

60 nm ultra-thin sections were cut and collected on gold EM grids and incubated with a primary antibody of either rabbit anti-GABA (Sigma) or rabbit anti-glutamate (Sigma). A secondary antibody goat anti-rabbit (BBI) conjugated to 15 nm gold particles were used at concentrations of 1:50 for GABA and 1:25 for glutamate.

Data Analysis

The gold density for all ChAT immunoperoxidase terminals was calculated and used to determine the co- existence of GABA or glutamate. "

Results

Graphs showed frequency distribution in histograms illustrating the relative density of gold particles associated with double labeled GABA/glutamate in cholinergic terminals in the central median (CM) parafasicular (PF), parafasicular dorsal lateral (Pf-dl), Lateral geniculate nucleus (LGen,) and ventral posterolateral thalamic nucleus (VPL). The average surface density of gold particles (± SEM) for each set of terminals was provided. Negative controls for GABA immunostaining are putative glutamatergic terminals forming asymmetric synapses. Negative controls for glutamate are putative GABAergic terminals forming symmetric synapses.

Conclusions and Future Studies

GABA does indeed co-exist within cholinergic terminals in intralaminar nuclei in the thalamus and not relay nuclei. Preliminary data suggests that there is a higher degree of co-existence of glutamate with acetylcholine in intralaminar nuclei than relay nuclei. Further experimentation is required to substantiate these findings.

Further Studies

• Elucidate the mechanisms of co-transmitter release.
• Determine the post-synaptic effects of the co-release of neurotransmitters on thalamic neuronal activity.

Acknowledgements and Funding Attributions

A special thanks to Mrs. Susan Maxson and Mr. Dinesh Raju for technical assistance. Pat Marstellar and Cathy Quinones for extensive organization effort on behalf of the SURE program. This study is supported by grants from the U.S. National Institute of Health. Grant number: R01NS37948-01, Grant number RR00165, and the HHMI for funding the SURE program at Emory University.