Electrophysiological properties of catecholaminergic neurons in the norepinephrine-deficient mouse

Abstract

To determine how norepinephrine affects the basic physiological properties of catecholaminergic neurons, brain slices containing the substantia nigra pars compacta and locus coeruleus were studied with cell-attached and whole-cell recordings in control and dopamine beta-hydroxylase knockout (Dbh -/-) mice that lack norepinephrine. In the cell-attached configuration, the spontaneous firing rate and pattern of locus coeruleus neurons recorded from Dbh -/- mice were the same as the firing rate and pattern recorded from heterozygous littermates (Dbh +/-). During whole-cell recordings, synaptic stimulation produced an alpha-2 receptor-mediated outward current in the locus coeruleus of control mice that was absent in Dbh -/- mice. Normal alpha-2 mediated outward currents were restored in Dbh -/- slices after pre-incubation with norepinephrine. Locus coeruleus neurons also displayed similar changes in holding current in response to bath application of norepinephrine, UK 14304, and methionine-enkephalin. Dopamine neurons recorded in the substantia nigra pars compacta similarly showed no differences between slices harvested from Dbh -/- and control mice. These results indicate that endogenous norepinephrine is not necessary for the expression of catecholaminergic neuron firing properties or responses to direct agonists, but is necessary for auto-inhibition mediated by indirect alpha-2 receptor stimulation.

Figure 1

The firing pattern and membrane conductances in vitro are the same between Dbh −/− and control (Dbh +/−) mice. (A) Cell-attached voltage recording of a noradrenergic neuron firing rate and pattern in a slice containing the locus coeruleus from a Dbh −/− mouse (left) and a control mouse (right). (B) No differences were observed between Dbh −/− (n=16) and control mice (n=18). (C) The current-voltage relationships of Dbh −/− (n=21) and control (n=24) mice are indistinguishable. (D) Currents evoked from noradrenergic neurons in response to voltage steps from −50 to −90 mV (holding current = −60 mV) in a Dbh −/− mouse (left) and a control mouse (right).

Figure 2

Response of LC neurons to cocaine is the same between Dbh −/− and control mice. (A) Synaptic stimulation of noradrenergic receptors (Electrical Stim.) and iontophoretic application of NE (Iontophoresis) during a voltage clamp recording of an LC neuron obtained from a Dbh +/− mouse. Bath application of cocaine (3μM) induces an increase in the peak amplitude and duration of the outward current (“cocaine” black trace). The outward current is blocked by bath application of the α-2 antagonist, yohimbine (10μM; “yohimbine” black trace). (B) Before iontophoretic application of NE, electrical stimulation of the slice results in no response of the recorded neuron obtained from a Dbh −/− mouse. However, after the third transient application of NE iontophoresis (50 ms application, once per minute), electrical stimulation of the slice produces a synaptically-induced outward current (“3 min” red trace). After the sixth iontophoretic application of NE, the outward current induced by electrical stimulation has reached its maximum amplitude (“6 min” black trace). (C) Summarized data showing that cocaine induces the same increase in peak amplitude of the outward current induced by synaptic stimulation in Dbh −/− and control mice.

Figure 3

Response of LC neurons to α-2-adrenerigc and μ-opioid receptor stimulation is the same between Dbh −/− and control mice. (A) Current recording from a Dbh −/−mouse. Bath application of norepinephrine (NE, 30μM) induces an outward current that washes out. The α-2-adrenergic agonist UK 14304 (UK, 100 nM) induces an outward current that is blocked by the α-2-adrenergic antagonist, yohimbine (Yoh, 10μM). The μ-opioid agonist, methionine-enkepahlin (Met-Enk, 10μM), also induced an outward current (single trace). (B) Summarized data. No difference was observed in the outward currents induced by bath application of any agent tested.

Figure 4

D2 dopamine receptor sensitivity on DA neurons is unchanged in Dbh −/− mice. The DA sensitivity was assessed by measuring D2 autoreceptor-mediated currents from DA neurons of the substantia nigra pars compacta (SNC). The maximal DA response was determined with a 5 second iontophoretic application (+190 nA) of 1M DA (Ionto). Subsequently, neurons from Dbh −/− mice (A) and control mice (B) exhibited similar sensitivity to bath perfusion of DA (3-100 μM). Analysis of the summary data (C) indicated that there was no significant main effect of genotype or genotype-concentration interaction.