Medial septal GABAergic neurons express the somatostatin sst2A receptor: functional consequences on unit firing and hippocampal theta

Abstract

GABAergic septohippocampal neurons play a major role in the generation of hippocampal theta rhythm, but modulatory factors intervening in this function are poorly documented. The neuropeptide somatostatin (SST) may be one of these factors, because nearly all hippocampal GABAergic neurons projecting to the medial septum/diagonal band of Broca (MS-DB) express SST. In this study, we took advantage of the high and selective expression of the SST receptor sst2A in MS-DB to examine its possible role on theta-related activity. Immunohistochemical experiments demonstrated that sst2A receptors were selectively targeted to the somatodendritic domain of neurons expressing the GABAergic marker GAD67 but were not expressed by cholinergic neurons. In addition, a subpopulation of GABAergic septohippocampal projecting neurons expressing parvalbumin (PV) also displayed sst2A receptors. Using in vivo juxtacellular recording and labeling with neurobiotin, we showed that a number of bursting and nonbursting neurons exhibiting high discharge rates and brief spikes were immunoreactive for PV or GAD67 and expressed the sst2A receptor. Microiontophoresis applications of SST and the sst2A agonist octreotide (OCT) showed that sst2A receptor activation decreased the discharge rate of both nonbursting and bursting MS-DB neurons and lessened the rhythmic activity of the latter. Finally, intraseptal injections of OCT and SST in freely moving rats reduced the power of hippocampal EEG in the theta band. Together, these in vivo experiments suggest that SST action on MS-DB GABAergic neurons, through sst2A receptors, represents an important modulatory mechanism in the control of theta activity.

Figure 1.

Regional and cellular localization of the sst_2A receptor in the rat MS-DB complex as visualized by immunohistochemistry. A, Schematic representation of a frontal section (interaural 9.48) illustrating the localization of the MS-DB. The red frame depicts the region of the MS-DB shown in B. B, Low-power magnification of sst_2A-immunoreactive cells using chromogenic labeling. Note that numerous neuronal cell bodies and processes are distributed throughout the dorsoventral extent of the nucleus. C, D, Representative examples of the numerous sst_2A-positive fusiform cells found in the MS-DB using fluorescence labeling. E, Albeit less numerous, immunoreactive cells for the sst_2A receptor also displayed a polygonal shape. F-H, As evidenced in confocal single-optical sections (false inverted black and white colors), sst_2A receptor immunoreactivity decorates the plasmic membrane of somata (F), proximal dendrites (F), and distal processes of medium (G) and small (H) sizes. I-K, Processes immunoreactive for the sst_2A receptor (sst_2A; I) also express MAP2 immunoreactivity (MAP2; J) as evidenced by the yellow signal observed in the overlay panel (Overlay; K). L-N, In sections double labeled for sst_2A (L) and synaptophysin (SYN; M), no colocalization is observed between the two markers (N). Note, however, that SYN-immunoreactive elements are closely apposed to both sst_2A-immunoreactive elongated and cross-sectioned (L-N, boxes) processes (N, arrows). Scale bars: B, 100 μm; C-E, 10 μm; (in K) F-K, 4 μm; (in N) L-N, 2 μm. CPu, Caudate-putamen; Cx, cerebral cortex; LSD, dorsolateral septum; LV, lateral ventricle.

Figure 2.

Neurochemical characterization of sst_2A-expressing cells in the MS. A, Sections double labeled with the sst_2A receptor (sst_2A; red) and GAD67 (green) reveal that all sst_2A-immunoreactive cells also display GAD67 immunoreactivity. GAD67-positive cells do not all display sst_2A receptors (stars in top row). B, Low (top row) and high (bottom row) magnifications of confocal microscopic images of double-labeled sections for sst_2A (red) and ChAT (green) immunoreactivities demonstrate that both markers are not expressed by the same cells. C, Sections double labeled with the sst_2A receptor (red) and parvalbumin (PV; green) reveal that a subpopulation of sst_2A-immunoreactive cells also expresses PV (top row, arrows), as also illustrated at a higher magnification in the bottom row. D, Confocal images of triple-labeled sections with sst_2A (red), PV (blue), and somatostatin (SST; green) illustrate that numerous SST-immunoreactive axon-like terminals are located in close apposition with the somatodendritic domain of sst_2A/PV neurons (overlay, arrowheads). Scale bars: A, 10 μm; B, 40 μm (top row), 8 μm (bottom row); C, 40 μm (top row), 4 μm (bottom row); D, 8 μm.

Figure 3.

Immunohistochemical identification and electrophysiological characterization of a single juxtacellular labeled PV/sst_2A neuron in the rat MS-DB. A, Single confocal section showing a recorded neuron labeled with neurobiotin (red). This neuron was also immunoreactive for parvalbumin (B, blue) and sst_2A receptors (C, green) as illustrated in D. E, Extracellular recording from the neurobiotin-labeled neuron shown in A-D. The discharge profile is characterized by a theta-related bursting activity (4.0 Hz) and a high discharge rate (45 spike/s). F, Expanded scale from E indicating the short duration of the spike (0.38 ms). Scale bar: (in D) A-D, 10 μm.

Figure 4.

Immunohistochemical identification and electrophysiological characterization of a single juxtacellular labeled GAD67/sst_2A neuron in the rat MS-DB. A, Single confocal section showing a recorded neuron labeled with neurobiotin (red). This neuron was also immunoreactive for GAD67 (B, green) and sst_2A receptors (C, blue) as illustrated in D. E, Extracellular recording from the neurobiotin-labeled neuron shown in A-D. The discharge profile is characterized by a theta-related bursting activity (4.5 Hz) and a high discharge rate (24 spike/s). F, Expanded scale from E indicating the short duration of the spike (0.39 ms). Scale bar: (in D) A-D, 10 μm.

Figure 5.

Illustration of the effects of iontophoretic OCT application on the firing pattern of a bursting neuron in the MS-DB. A, During application of OCT (80 nA, 25 s), the discharge rate decreases by 57%. B, C, RB activity is impaired (C, expanded time scale corresponding to the stars in B). Bursts, composed of a reduced number of spikes, are less numerous. Note that the spike amplitude is increased, suggesting a hyperpolarizing effect of OCT. D, Autocorrelation histograms of representative 10 s periods of unit activity before (left), during (middle), and after (right) OCT application. Rhythmically bursting activity is considerably reduced but not completely abolished as shown by the residual peaks in the middle autocorrelation histogram.

Figure 6.

Illustration of the effects of iontophoretic SST application on the firing pattern of a bursting neuron in the MS-DB. A, During application of SST (80 nA, 20 s), the discharge rate decreases by 70%. B, C, RB activity is impaired (C, expanded timescale corresponding to the stars in B). Bursts, composed of a reduced number of spikes, are less numerous. D, Autocorrelation histograms of representative 10 s periods of unit activity before (left), during (middle), and after (right) OCT application. Rhythmically bursting activity is considerably reduced but not completely abolished as shown by the residual peaks in the middle autocorrelation histogram.

Figure 7.

Illustration of the inhibitory effect of SST on the firing pattern of a nonbursting neuron in MS-DB. Note that the discharge rate decreases by 70% during iontophoretic application of SST (80 nA, 40 s) and recovers progressively after application.

Figure 8.

Illustration of the effect of intraseptal OCT infusion on hippocampal theta in a freely moving rat. A, The relative power spectrum of hippocampal EEG in control condition is 10% in the 1-6 Hz band and 71% in the 6-9 Hz (theta) band. B, Twenty minutes after intraseptal infusion of OCT (0.5 nmol), the relative power increases to 43% in the 1-6 Hz band and decreases to 38% in the theta band (6-9 Hz). Note that the theta rhythm is less steady after OCT (sample in B).

Figure 9.

Time course of changes in hippocampal EEG after intraseptal OCT (A) or SST (B) infusions. Values are the mean ± SEM percentage of the power in the 1-6 and 6-9 Hz ranges relative to the total power. For both OCT and SST, note the opposite variations of the relative power between 1-6 and 6-9 Hz frequencies (top and bottom, respectively). Note the absence of effect of saline, the larger effect of the higher doses, and the progressive return to control values (^*p < 0.05; ^**p < 0.01; ^***p < 0.001).