Modulation of cognition and neuronal plasticity in gain- and loss-of-function mouse models of the schizophrenia risk gene Tcf4.

The transcription factor TCF4 was confirmed in several large genome-wide association studies as one of the most significant schizophrenia (SZ) susceptibility genes. Transgenic mice moderately overexpressing Tcf4 in forebrain (Tcf4tg) display deficits in fear memory and sensorimotor gating. As second hit, we exposed Tcf4tg animals to isolation rearing (IR), chronic social defeat (SD), enriched environment (EE), or handling control (HC) conditions and examined mice with heterozygous deletion of the exon 4 (Tcf4Ex4δ+/-) to unravel gene-dosage effects. We applied multivariate statistics for behavioral profiling and demonstrate that IR and SD cause strong cognitive deficits of Tcf4tg mice, whereas EE masked the genetic vulnerability. We observed enhanced long-term depression in Tcf4tg mice and enhanced long-term potentiation in Tcf4Ex4δ+/- mice indicating specific gene-dosage effects. Tcf4tg mice showed higher density of immature spines during development as assessed by STED nanoscopy and proteomic analyses of synaptosomes revealed concurrently increased levels of proteins involved in synaptic function and metabolic pathways. We conclude that environmental stress and Tcf4 misexpression precipitate cognitive deficits in 2-hit mouse models of relevance for schizophrenia.

In vivo recording of nerve conduction velocity of spinal CNS fibers in the mouse.

Anesthetic and surgical procedures and an electrophysiological method were developed for recording nerve conduction velocity (NCV) of CNS fibers in the murine spinal cord. Under intravenous anesthesia and artificial ventilation the lumbar spinal cord segments L1 to L4 and dorsal roots L3 to L5 on the left side were exposed by laminectomy. After stimulation of the dorsal root L4, a compound action potential (CAP) was recorded at the ipsilateral left fasciculus gracilis at the spinal cord level L1. The latency from stimulation to the CAP together with the measured distance between the electrodes was used for the determination of the NCV. NCV of the fastest fibers in the fasciculus gracilis was observed to be approximately 28 m/s. Reversible decrease of the NCV was measured, in vivo, under general hypothermia. The technique described serves for in vivo electrophysiological investigations of spinal central fibers in wildtype and mutant mice.

Two-photon laser-scanning microscopy for single and repetitive imaging of dorsal and lateral spinal white matter in vivo.

We developed appropriate surgical procedures for single and repetitive multi-photon imaging of spinal cord in vivo. By intravenous anesthesia, artificial ventilation and laminectomy, acute experiments were performed in the dorsal and lateral white matter. By volatile anesthesia and minimal-invasive surgery, chronic repetitive imaging up to 8 months were performed in the dorsal column through the window between two adjacent spines. Transgenic mouse technology enabled simultaneous imaging of labeled axons, astrocytes and microglia. Repetitive imaging showed positional shifts of microglia over time. These techniques serve for investigations of cellular dynamics and cell-cell interactions in intact and pathologically changed spinal tissue.

Long lasting activity of nociceptive muscular afferents facilitates bilateral flexion reflex pattern in the feline spinal cord.

Chronic muscular limb pain requires the adoption of motor patterns distinct from the classic ipsilateral flexion, crossed extension and corresponding reciprocal inhibitions to acute exteroceptive stimulation. Using selective chemical activation of group III/IV afferents in gastrocnemius-soleus (GS) muscles we investigated bilaterally their reflex responses conditioned by (a) acute 'myositis' induced by intramuscular carrageenan; and (b) sub-acute 'myositis' induced by infusion of complete Freund's adjuvant (CFA). Reflex transmission was detected by monosynaptic testing and c-fos staining used to identify increased neuronal activity. In all control experiments with chemical stimulation of group III/IV afferents, ipsilateral responses conformed to the flexor reflex pattern. However, the expected contralateral facilitation of GS motoneurones occurred in fewer than 50% trials while only 9% of trials induced contralateral inhibition of flexor posterior-biceps-semitendinosus (PBSt) motoneurones. During carrageenan acute myositis contralateral PBSt was transiently facilitated by selective activation of group III/IV afferents. During CFA-induced myositis, contralateral only inhibition of GS motoneurones occurred instead of any facilitation, while bidirectionally a crossed facilitation of PBST dominated. These reflex changes were mirrored in an enhanced number of neurones with enhanced c-fos expression. Muscle pain, particularly if chronically persistent, requires another behavioural response pattern than acute exteroceptive pain.

Heterologous expression of a glial Kir channel (KCNJ10) in a neuroblastoma spinal cord (NSC-34) cell line.

Heterologous expression of Kir channels offers a tool to modulate excitability of neurons which provide insight into Kir channel functions in general. Inwardly-rectifying K+ channels (Kir channels) are potential candidate proteins to hyperpolarize neuronal cell membranes. However, heterologous expression of inwardly-rectifying K+ channels has previously proven to be difficult. This was mainly due to a high toxicity of the respective Kir channel expression. We investigated the putative role of a predominantly glial-expressed, weakly rectifying Kir channel (Kir4.1 channel subunit; KCNJ10) in modulating electrophysiological properties of a motoneuron-like cell culture (NSC-34). Transfection procedures using an EGFP-tagged Kir4.1 protein in this study proved to have no toxic effects on NSC-34 cells. Using whole cell-voltage clamp, a substantial increase of inward rectifying K+ currents as well as hyperpolarization of the cell membrane was observed in Kir4.1-transfected cells. Na+ inward currents, observed in NSC-34 controls, were absent in Kir4.1/EGFP motoneuronal cells. The Kir4.1-transfection did not influence the NaV1.6 sodium channel expression. This study demonstrates the general feasibility of a heterologous expression of a weakly inward-rectifying K+ channel (Kir4.1 subunit) and shows that in vitro overexpression of Kir4.1 shifts electrophysiological properties of neuronal cells to a more glial-like phenotype and may therefore be a candidate tool to dampen excitability of neurons in experimental paradigms.

In vivo measurement of conduction velocities in afferent and efferent nerve fibre groups in mice.

Electrophysiological investigations in mice, particularly with altered myelination, require reference data of the nerve conduction velocity (CV). CVs of different fibre groups were determined in the hindlimb of anaesthetized adult mice. Differentiation between afferent and efferent fibres was performed by recording at dorsal roots and stimulating at ventral roots, respectively. Correspondingly, recording or stimulation was performed at peripheral hindlimb nerves. Stimulation was performed with graded strength to differentiate between fibre groups. CVs of the same fibre groups were different in different nerves of the hindlimb. CVs for motor fibres were for the tibial nerve (Tib) 38.5±4.0 m/s (Agamma: 16.7±3.0 m/s), the sural nerve (Sur) 39.3±3.1 m/s (12.0±0.8 m/s) and the common peroneal nerve (Per) 46.7±4.7 m/s (22.2±4.4 m/s). CVs for group I afferents were 47.4±3.1 m/s (Tib), 43.8±3.8 m/s (Sur), 55.2±6.1 m/s (Per) and 42.9±4.3 m/s for the posterior biceps (PB). CVs of higher threshold afferents, presumably muscle and cutaneous, cover a broad range and do not really exhibit nerve specific differences. Ranges are for group II 22-38 m/s, for group III 9-19 m/s, and for group IV 0.8-0.9 m/s. Incontrovertible evidence was found for the presence of motor fibres in the sural nerve. The results are useful as references for further electrophysiological investigations particularly in genetically modified mice with myelination changes.

Role of L-DOPA in spinal nociceptive reflex activity: higher sensitivity of Aδ versus C fibre-evoked nociceptive reflexes to L-DOPA.

The role of L-DOPA in spinal nociceptive reflex activity has been re-evaluated. In high spinal cats, with supraspinal loops being excluded, the onset of reflex facilitation induced by noxious radiant heat is delayed after injection of L-DOPA by 4 to 10 s, i.e. the early component of nociceptive reflex facilitation is blocked, while the late component persisted. Further investigations have shown that the early component of reflex facilitation induced by noxious radiant heat is mediated by Adelta-fibres and the late component by C-fibres. Therefore, it can be assumed that L-DOPA, like opioids, preferentially blocks the transmission in nociceptive reflex pathways from Adelta-fibres.