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Dopaminergic signalling in nucleus incertus – unexpected inhibitory and excitatory effects of D2 activation

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Szlaga A., Gugula A., Sambak P., Blasiak A.

Acta Neurobiologiae Experimentalis

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2018

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tom 78

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nr Suppl.1

Nucleus incertus (NI) is a brainstem structure involved in stress response, arousal, and food intake control. NI is a main source of relaxin-3 in the brain, and relaxin-3 was shown to alter reward and stress related behaviours. Re‑ cently, dopamine receptors were identified in the NI, how‑ ever the effect of their activation on NI neuronal activity was unknown. Therefore, the current study aimed to char‑ acterise the effect of dopamine receptor activation on NI neuronal activity, as well as identify the source of tyrosine hydroxylase (TH)-positive fibres in the structure. Wholecell patch-clamp recordings were used to assess the re‑ sponsiveness of NI neurons to selective D1R and D2R ag‑ onist application. Track-tracing combined with anti-TH immunohistochemical staining was used to define the source of TH immunoreactive fibres in the NI. D1R ago‑ nist SKF-81297 (10 µM) caused depolarization of NI cells by 5.01±0.75 mV (mean change ± SEM). Depolarization persist‑ ed in the presence of tetrodotoxin and glutamate/GABA receptors antagonists which indicates direct postsynaptic action of SKF-81297. Interestingly, activation of D2Rs with quinpirole (20 µM) induced both inhibitory and excitato‑ ry effects on NI neuron activity. In 56% of NI neurons an outward current (13.87±6.55 pA), decrease in action poten‑ tial firing frequency (3.47±1.33 Hz), and hyperpolarization of quiescent cells (2.16±1.01 mV) was observed after quin‑ pirole administration. In 28% of the neurons an increase in inward current amplitude (13.73±2.08 pA) and increase in frequency of action potentials (0.75±0.37 Hz) was recorded after quinpirole application. Both excitatory and inhibitory action of quinpirole persisted in the presence of tetrodo‑ toxin and GABA/glutamate receptors antagonists. Results of track-tracing experiments allowed identification of A11 and A13 cell groups as a source of dopamine innervation in the NI. D1R and D2R are localised postsynaptically on NI neurons. Surprisingly, D2R activation exerted both direct inhibitory and excitatory effects on NI neurons, suggesting a diverse action for dopamine receptor agonists on neuro‑ chemically and/or functionally distinct cell classes in this structure. Identification of A11 and A13 dopamine cells groups as a potential source of TH immunoreactive fibres in the NI allows us to conclude that dopaminergic inner‑ vation of the NI may be involved in the control of alertness and sensorimotor response to salient stimuli.

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Maternal separation modifies dendritic spine density in ventral tegmental area neurons in a sub-region-specific manner: study in female rats

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Celary A., Gugula A., Tylko G., Hess G., Blasiak A.

Acta Neurobiologiae Experimentalis

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2019

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tom 79

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nr Suppl.1

Early life stress disrupts development of the human and animal brain and increases the risk of psychophysiological disorders and susceptibility to addiction in adulthood. Maternal separation (MS), an animal model of early life stress, was shown to raise predisposition to addictive behaviours and change neuronal activity as well as dendritic spine density in a range of brain structures. Mesocorticolimbic dopaminergic pathways originating from the ventral tegmental area (VTA) play a crucial role in the development of addiction, however, the influence of MS on VTA neuronal architecture remains obscure. AIM(S): The current study aimed to verify the influence of MS on VTA neuronal dendritic spine density, a possible anatomical substrate of functional changes in the ascending dopaminergic pathways. METHOD(S): Female rat pups were separated from dams for 3 hours daily from PND2 to 14. At PND65, rats were decapitated, Golgi-Cox staining was performed, and the density of spines was calculated manually on I‑III‑ order dendrites. Given the functional and anatomical heterogeneity of the VTA, analyzed neurons were assigned to specified VTA sub‑regions. RESULTS: In rats subjected to MS, significantly lower density of dendritic spines on neurons in ventromedial (II‑order branches – 15%), dorsolateral (II‑ and III‑order segments – 24 – 23% respectively), and dorsomedial (I‑order branches – 25%) VTA, was observed when compared to control. No significant changes in spine density were observed in ventrolateral VTA (only 6% decrease in spine density). CONCLUSIONS: The observed decrease in dendritic spine density in VTA neurons can be linked to a reduced number of excitatory synapses that may underlie altered activity of mesocorticolimbic pathways, altered dopamine release, and increased susceptibility to addictions observed after MS and childhood trauma. Sub‑region specificity of observed changes points to varied sensitivity of VTA neurons to stress. FINANCIAL SUPPORT: Funding: NSC-Poland UMO‑2016/21/B/NZ4/00204.

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Maternal separation stress alters excitability and stress induced c-fos expression in ventral tegmental area dopaminergic neurons of adult female rats

88%

Gugula A., Spyrka J., Cizio K., Hess G., Blasiak A.

Acta Neurobiologiae Experimentalis

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2019

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tom 79

|

nr Suppl.1

INTRODUCTION: Traumatic experiences in childhood and maternal separation (MS) in rodents disrupts proper development of the brain including dopaminergic (DA) mesocorticolimbic pathways originating from ventral tegmental area (VTA). Importantly, early‑life stress predisposes for neuropsychiatric disorders and addiction in adulthood. Moreover, MS stress increases the number of VTA tyrosine hydroxylase (TH) immunoreactive DA neurons, raises baseline dopamine levels, and increases its release in response to acute stress in adult rats. However, neuronal mechanisms of these changes have not been fully explored. AIM(S): The current study aimed at determining the influence of MS on VTA DA cells electrophysiology and responsiveness to acute stress at the level of c-fos expression. METHOD(S): Female rats were submitted to MS during PND 2‑14, 3 h daily. In adulthood, some of the rats were subjected to restraint stress and subsequently perfused. The VTA region was cut, stained against TH and c-fos, and double stained neurons were counted. Remaining animals were sacrificed and brain slices containing VTA were prepared for electrophysiological patch‑clamp experiments. Recorded biocytin‑filled cells were stained and assessed as TH+ or TH‑. RESULTS: Our data show that exposure to early life stress leads to a significant reduction of the rheobase and an increased number of action potentials generated vs. injected current – indices of neuronal excitability. Importantly, it was altered in both TH+ and TH‑ VTA neurons. MS combined with restraint stress significantly increased the number of dorsal but not ventral VTA TH+/c‑fos+ cells. CONCLUSIONS: Observed changes in excitability of VTA DA neurons may constitute a neuronal mechanism of the reported elevated dopamine release after MS. Our data indicate that MS alters reactivity of dorsal but not ventral VTA cells to acute stress, which suggests a greater raise in stress-induced DA release in structures innervated by the dorsal VTA. FINANCIAL SUPPORT: Funding: NSC-Poland UMO- ‑2016/21/B/NZ4/00204.

Ograniczanie wyników

3 Acta Neurobiologiae Experimentalis

3 Blasiak A.

3 Gugula A.

2 Hess G.

1 Celary A.

1 Cizio K.

1 Sambak P.

1 Spyrka J.

1 Szlaga A.

1 Tylko G.

2 2019

1 2018

Dopaminergic signalling in nucleus incertus – unexpected inhibitory and excitatory effects of D2 activation

Maternal separation modifies dendritic spine density in ventral tegmental area neurons in a sub-region-specific manner: study in female rats

Maternal separation stress alters excitability and stress induced c-fos expression in ventral tegmental area dopaminergic neurons of adult female rats