Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Summary

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Abstract

Long-lasting changes in the brain or ‘brain plasticity’ underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in ‘enriched environments’ (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of ‘DA’ genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson’s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

Introduction

DArgic signaling by neurons in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) of the midbrain is thought to be important for reward-motivated cognitive, emotive and motor behaviors. However, too much or too little midbrain DA signaling causes many disabling symptoms in a variety of neurological disorders (e.g. Parkinson’s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction). Drugs that increase or decrease DA signaling alleviate these symptoms, however they also produce side-effects attributable to dysregulated signaling and off-target effects. Drug efficacy also declines over time due to c....

Protocol

NOTE: All experimental procedures on animals were approved by the Florey Institute of Neuroscience & Mental Health Animal Ethics Committee and conform to Australia’s National Health and Medical Research Council published code of practice for the care and use of animals for scientific purposes (7^th edition, 2004).

1. Environmental Manipulations

1. Gender Pairing
   1. Use sexually mature (>8 week old), age-matched male and female mice.
      NOTE: We typically use C57BL/6 mice, but have had the same outcomes using Swiss mice in this protocol. We typically use n = 8 males and n = 8 females for each ex....

Results

Adult mice subjected to these environmental manipulations have altered numbers of midbrain (SNc and VTA), but not LC, TH+ neurons, and EE plus concurrent midbrain infusion of either picrotoxin or bicuculline (GABA_A receptor antagonists) abolishes EE-induction of more SNc TH+ neurons. These data were previously published in⁶. The present data were compiled in replicate experiments performed as part of that previous study, but have not been published elsewhere.

Specifically.......

Discussion

Environmental manipulations

The motivation behind the design of these environmental manipulations (gender pairing and environmental enrichment) was to determine whether the environment, and/or behavior prompted by the environment, is associated with changes in the number of midbrain DA neurons. The focus was therefore on providing environments and stimulating behaviors that are likely to engage midbrain DA signaling. These included pairing with the opposite gender, and environmental e.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Isofluorane	Baxter Healthcare Pty Ltd, Baxter Drive, NSW 2146, Australia	AHN3640
ALZET Osmotic pump 1002	DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530	0004317
ALZET Brain infusion kit 1	DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530	0004760
ALZET cannula holder 1	DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530	0008860
Vertex Monomer Self-curing (dental acrylic solvent)	Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands	n/a
Vertex Self Curing (dental acrylic powder)	Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands	n/a
METACAM (Meloxicam)	Troy Laboratories, 98 long Street, smithfield NSW 2164 Australia	L10100
Sodium Pentobarbitone	Lethabarb, Virbac, Milperra, NSW, Australia	571177
Normal goat serum	chemicon-temecula, CA	S26-Litre
Triton X-100	Merck Millipore Headquarters , 290 Concord road, Billerica, MA 01821	1.08603.1000
Polyclonal rabbit anti-tyrosine hydroxylase	Merck Millipore Headquarters , 290 Concord road, Billerica, MA 01821	AB152
Polyclonal biotinylated goat anti-rabbit	Dako Australia Pty. Ltd., Suite 4, Level 4, 56 Berry street, North Sydney, NSW, Australia 2060	EO432
Avidin peroxidase	Sigma-aldrich, Castle Hill, NSW 1765 AU	A3151-1mg
Diamino-benzidine	Sigma-aldrich, Castle Hill, NSW 1765 AU	D-5637
Stereo Investigator	MicroBrightField Bioscience, 185 Allen Brook Lane, Suite 101, Williston, VT 05495	n/a