Silke Patz

Neuronal activity and neurotrophic factors regulate GAD‐65/67 mRNA and protein expression in organotypic cultures of rat visual cortex

Environmental factors are known to regulate the molecular differentiation of neocortical interneurons. Their class‐defining transmitter synthetic enzymes are the glutamic acid decarboxylases (GAD); yet, fairly little is known about the developmental regulation of transcription and translation of the GAD‐65/67 isoforms. We have characterized the role of neuronal activity, neurotrophins and afferent systems for GAD‐65/67 expression in visual cortex in organotypic cultures (OTC) compared with in vivo in order to identify cortex‐intrinsic regulatory mechanisms. Spontaneously active OTC prepared at postnatal day 0 displayed from 10 days in vitro (DIV) onwards 12–14% GAD‐65/GAD‐67 neurons similar to in vivo. However, GAD‐65 mRNA was higher, whereas GAD‐67 protein was lower, than in vivo. During the first week neurotrophins increased whereas the Trk receptor inhibitor K252a and MEK inhibitors decreased both GAD mRNAs and proteins. After 10 DIV GAD expression no longer depended on neurotrophin signalling. Activity‐deprived OTC revealed only 6% GAD‐67 neurons and mRNA and protein were reduced by 50%. GAD‐65 mRNA was less reduced, but protein was reduced by half, suggesting translational regulation. Upon recovery of activity GAD mRNAs, cell numbers, and both proteins quickly returned to normal and these ‘adult’ levels were resistant to late‐onset deprivation. In 20 DIV activity‐deprived OTC, only neurotrophin 4 increased GAD‐65/67 mRNAs, rescued the percentage of GAD‐67 neurons and increased both proteins in a TrkB‐dependent manner. Activity deprivation had thus shifted the period of neurotrophin sensitivity to older ages. The results suggested neuronal activity as a major regulator differentially affecting transcription and translation of the GAD isoforms. The early presence of neuronal activity promoted the GAD expression in OTC to a neurotrophin‐independent state suggesting that neurotrophins play a context‐dependent role.

Neurotrophins induce short-term and long-term changes of cortical neurotrophin expression

Neuronal activity, hormones, transmitters, physical exercise and enrichment influence cortical neurotrophin expression. Neurotrophins then elicit structural and physiological changes, and regulate gene expression. This prompted the hypothesis that neurotrophins themselves are involved in regulating neurotrophin expression. Here we investigated the mRNA expression level of brain‐derived neurotrophic factor (BDNF), neurotrophin‐4 (NT‐4), NT‐3 and nerve growth factor (NGF) as well as the tyrosine receptor kinases TrkB and TrkC receptor in response to BDNF, NT‐4, NT‐3 and NGF pulses in organotypic cortex cultures. Single neurotrophin pulses evoked a dramatic up‐ or down‐regulation of some, but not all four, neurotrophin mRNAs, even within 3–24 h, indicating an immediate impact on neurotrophin transcription. Most strikingly, neurotrophin pulses during the first 10 days in vitro (DIV) potentiated the expression of some neurotrophin mRNAs at 20 DIV, suggesting that early trophic factor experience influences the expression levels seen later in development. The NT‐3 mRNA expression, for example, was consistently promoted by NGF and BDNF, suggesting that these two factors help to maintain the low level of NT‐3 found in adult cortex. Rapid bidirectional changes characterized the NT‐4 mRNA expression. A single pulse of NT‐4 transiently increased NT‐4 mRNA, whereas a BDNF pulse transiently reduced NT‐4 mRNA. Surprisingly, NGF strongly potentiated BDNF mRNA and in particular NT‐4 mRNA. By contrast, TrkB mRNA remained constant at ages or time points at which other mRNAs amplified from the very same cDNA libraries revealed dramatic increases or decreases. Our study suggests the existence of a complex regulatory neurotrophin network controlling the expression of other neurotrophins.

Developmental changes of neurotrophin mRNA expression in the layers of rat visual cortex.

Neurotrophins are essential factors for the structural, neurochemical and functional maturation of the brain including developmental and adult plasticity. Northern blots and polymerase chain reaction revealed the expression of neurotrophin 4 (NT4), neurotrophin 3 (NT3), brain‐derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the cortex. The cellular producers of NT3 and BDNF have been characterized by anatomical methods as being mostly pyramidal, and the tyrosine kinase B (TrkB) receptor is expressed by many cortical neurons. However, these methods have so far failed to identify the cells producing NT4 and NGF mRNA. These factors are much lower in expression than, e.g. BDNF, and apparently remain below detection levels of in situ hybridization. Given their specific actions on cell types and afferent systems, knowledge about the producing cell types is highly desirable. To narrow down on the producing cell types, we quantified by reverse transcriptase‐polymerase chain reaction (RT‐PCR) the developmental changes of BDNF, NT3, NT4, NGF and TrkB mRNA expression in total visual cortex lysates, and in the cortical layers dissected by tangential cryostat sectioning. We found dramatic changes in laminar expression of NT3 and NGF, mild changes of NT4, and no changes of BDNF and TrkB mRNA. For instance, NT3 is important early on for thalamocortical axons, and we found transient peaks of NT3 mRNA expression first in layer VI, then in layer IV. NT4 mRNA was in layers IV and VI, suggesting NT4 protein production in thalamorecipient layers, but peak expression gradually shifted to upper layers as did NGF expression. The layer‐specific developmental expression shifts of neurotrophin mRNAs correlate with morphogenetic processes.

GABAC receptors are expressed in GABAergic and non-GABAergic neurons of the rat superior colliculus and visual cortex

GABAC receptors are enriched in the upper grey layers of the mammalian superior colliculus and contribute to synaptic processing. Electrophysiological data suggested that the GABAC receptor ρ subunits are expressed by GABAergic interneurons which represent about half of the neurons in the stratum griseum superficiale (SGS). Combining in situ hybridization for ρ2 receptor mRNA and the glutamic acid decarboxylase GAD-65 mRNA confirmed this assumption. A majority of ρ-labeled neurons in SGS and pretectum are GABAergic. Combining in situ hybridization with immunohistochemistry for the two projection neuron markers calbindin and parvalbumin revealed that a few ρ2 mRNA expressing cells coexpressed calbindin, but not parvalbumin. In visual cortex, ρ2 mRNA was present in pyramidal neurons and parvalbumin-containing interneurons. The results show that in the SGS primarily GABAergic neurons express GABAC receptors whereas the majority of tectothalamic calbindin neurons and intrinsically projecting parvalbumin neurons do not.

Leukemia inhibitory factor impairs structural and neurochemical development of rat visual cortex in vivo.

&NA; Minipump infusions into visual cortex in vivo at the onset of the critical period have revealed that the proinflammatory cytokine leukemia inhibitory factor (LIF) delays the maturation of thalamocortical projection neurons of the lateral geniculate nucleus, and tecto‐thalamic projection neurons of the superior colliculus, and cortical layer IV spiny stellates and layer VI pyramidal neurons. Here, we report that P12–20 LIF infusion inhibits somatic maturation of pyramidal neurons and of all interneuron types in vivo. Likewise, DIV 12–20 LIF treatment in organotypic cultures prevents somatic growth GABA‐ergic neurons. Further, while NPY expression is increased in the LIF‐infused hemispheres, the expression of parvalbumin mRNA and protein, Kv3.1 mRNA, calbindin D‐28k protein, and GAD‐65 mRNA, but not of GAD‐67 mRNA or calretinin protein is substantially reduced. Also, LIF treatment decreases parvalbumin, Kv3.1, Kv3.2 and GAD‐65, but not GAD‐67 mRNA expression in OTC. Developing cortical neurons are known to depend on neurotrophins. Indeed, LIF alters neurotrophin mRNA expression, and prevents the growth promoting action of neurotophin‐4 in GABA‐ergic neurons. The results imply that LIF, by altering neurotrophin expression and/or signaling, could counteract neurotrophin‐dependent growth and neurochemical differentiation of cortical neurons. HighlightsLIF inhibits somatic growth of cortical pyramidal neurons and interneurons in vivo.LIF attenuates developmental upregulation of key interneuronal marker proteins.LIF modulates neurotrophin expression.LIF seems to counteract neurotrophin‐dependent neuronal differentiation.

GABAC receptor subunit mRNA expression in the rat superior colliculus is regulated by calcium channels, neurotrophins, and GABAC receptor activity

The distribution of mRNA for the ρ2 subunit of the GABAC receptor is much broader in organotypic SC cultures than in vivo, suggesting that GABAC receptor expression is regulated by environmental factors. Electrophysiological recordings indicate that neurons in SC cultures have functional GABAC receptors, although these receptors exhibited smaller conductance than in vivo, probably due to increased ρ2 subunit expression. Adding cortical input, treatment with various neuromodulators, and blocking neuronal activity with TTX failed to affect the expression of ρ2 subunits. Electrophysiological recordings revealed the presence of spontaneous Ca2+ currents in SC cultures and preventing these, by treatment with blockers of L-type Ca2+ channels, caused ρ2 mRNA expression to decline to in vivo levels. In contrast, ρ1 subunit mRNA levels remained unchanged, indicating that the two subunits are independently regulated. Surprisingly, both tonic activation and blockade of GABAC receptors upregulated ρ1/ρ2 mRNA expression. Further, NGF and BDNF promoted such expression during an early postnatal time window. In vivo, expression of the ρ2 mRNA in the SC, and the ρ2/ρ3 mRNA in the retina increased with age. Expression of the ρ2 mRNA in the visual cortex, and the ρ1 mRNA in the retina and SC was constant. Subunit mRNA expression was similar in dark-reared animals, indicating that visual experience has no influence. These experiments suggest that GABAC receptor expression in the SC is regulated during postnatal development. While visual experience seems to have no influence on GABAC receptor subunits, spontaneous calcium currents selectively promote ρ2 expression and both ρ1 and ρ2 are autoregulated both by GABAC receptor activity and by neurotrophic factors.

Interneuronal growth and expression of interneuronal markers in visual cortex of mice with transgenic activation of Ras

The synRas transgenic mice express constitutively activated Valin12-Harvey Ras in postnatal neocortical pyramidal neurons. This leads to somatodendritic hypertrophy, higher densities of spines and synapses, and an enhancement of synaptic long-term potentiation associated with an increased glutamate receptor-mediated activity. It was less clear how the interneurons respond to these alterations, and this prompted the quantitative assessment of interneuron neurochemistry. Interneurons rarely expressed the transgene, however, several interneuron types displayed a transient somatic hypertrophy. Furthermore, NPY mRNA expression was persistently increased as were the laminar percentages of labeled neurons. The expression of parvalbumin and voltage-gated potassium channels Kv3.1b/3.2 was unchanged. A significant decline of GAD-67, but not GAD-65, mRNA expressing neurons was observed in layer VI in animals older than P60. This suggested that subtle deficits in inhibition and enhanced excitation evoke the interneuronal changes in the synRas-transgenic mouse cortex.

Das Umfeld macht es: wie Interneurone fürs Leben lernen!

Zusammenfassung Die GABAergen inhibitorischen Interneurone der Hirnrinde bilden eine morphologisch, neurochemisch und elektrophysiologisch heterogene Klasse von Neuronen. Die Diversität ist nicht allein genetisch determiniert, sondern wird während der postnatalen Entwicklung durch Umgebungsfaktoren beeinflusst: bioelektrische Aktivität, Neurotrophine, Afferenzen und sensorische Erfahrung steuern die Entwicklung der neurochemischen Phänotypen. Wir haben im Modell der organtypischen Kultur zentraler visueller Strukturen die ontogenetische Expression von Glutamatdecarboxylasen-65/ 67 (GAD-65/67), Parvalbumin (PARV) und Neuropeptid Y (NPY) analysiert. Die Expression dieser drei Funktionsmarker ist von Aktivität und Neurotrophinen abhängig. Die PARV- und die NPY-Expression werden zudem durch thalamische Afferenzen moduliert. Die NPY-Expression muss von jungen Neuronen regelrecht „erlernt“ werden, um im adulten Kortex bedarfsabhängig aktiviert werden zu können. Wir postulieren die Existenz kritischer Perioden molekularer Plastizität in denen junge Interneurone in Abhängigkeit von Umgebungsfaktoren Phänotypentscheidungen treffen.

Interneuron synaptopathy in developing rat cortex induced by the pro-inflammatory cytokine LIF

ABSTRACT Pro‐inflammatory cytokines of the IL‐6 family have been linked to the etiology of epilepsy and mental disorders. After infusion of the IL‐6 family member leukemia inhibitory factor (LIF) into postnatal day 12–20 rat visual cortex (period of synaptogenesis; equals early childhood in human) and equivalent ages in organotypic cultures (OTC), somatic growth of cortical and subcortical neurons is delayed, expression of interneuron markers reduced and expression of neurotrophins transiently reduced. Further, LIF prevents NT4 from promoting soma growth of GABA‐ergic interneurons. We now show that LIF‐treatment from DIV 12–20 in OTC compromises the differentiation of fast‐spiking GABA‐ergic basket neurons: GAD‐65/67, Kv3.2, and synaptotagmin‐2 proteins are reduced, dendrites are shorter, and axonal varicosities are smaller. Bitufted and Martinotti interneurons are barely affected. Pyramidal cells display lower dendritic spine densities, more filopodia, and shorter axon initial segments. &bgr;IV‐spectrin, Cav3.1, GABAA receptor subunits &agr;1 and &agr;2, and the phosphorylation of GluN2B at Y1472 are reduced. The frequency of calcium events in pyramidal cells and interneurons is increased, and the antiepileptic neuropeptide Y is upregulated, suggesting a hyperexcitability of the network. In the presence of LIF, neurotrophins fail to activate MAP kinase phosphorylation and c‐fos expression, and exogenous NT4 fails to promote the maturation of pyramidal cells and interneurons as it normally would. After discontinuing LIF treatment, bouton size and expression levels of affected proteins normalize except for synaptotagmin‐2; moreover, hyperexcitability persists. The results suggest that elevated levels of inflammatory cytokines during this developmental period cause a lasting maldevelopment in particular of basket cells. HIGHLIGHTSLIF inhibits growth of dendrites, axon initial segments and synaptic boutons.LIF attenuates developmental upregulation of key synaptic proteins.LIF lastingly impairs Syt‐2 expression required for synchronous GABA release.LIF prevents neurotrophins from activating MAPK and c‐fos signaling.